88kDa tumorigenic growth factor and antagonists

ABSTRACT

The invention relates to products and methods for treating cancer and for diagnosing tumorigenicity and other diseases associated with alteration in GP88 expression or action. Antagonists to an 88 KDa autocrine growth and tumorigenicity stimulator are provided which inhibit its expression or biological activity. The antagonists include antisense oligonucleotides and antibodies.

FIELD OF THE INVENTION

[0001] This invention relates to cell biology, physiology and medicine,and concerns an 88 kDa glycoprotein growth factor (“GP88”) andcompositions and methods which affect the expression and biologicalactivity of GP88. These compositions and methods are useful fordiagnosis and treatment of diseases including cancer.

REFERENCES

[0002] Several publications are referenced herein by Arabic numeralswithin parenthesis. Full citations for these references may be found atthe end of the specification immediately preceding the claims.

BACKGROUND OF THE INVENTION

[0003] The proliferation and differentiation of cells in multicellularorganisms is subject to a highly regulated process (1). A distinguishingfeature of cancer cells is the absence of control over this process;proliferation and differentiation become deregulated resulting inuncontrolled growth. Significant research efforts have been directedtoward better understanding this difference between normal and tumorcells. One area of research focus is growth factors and, morespecifically, autocrine growth stimulation.

[0004] Growth factors are polypeptides which carry messages to cellsconcerning growth, differentiation, migration and gene expression (2).Typically, growth factors are produced in one cell and act on anothercell to stimulate proliferation. However, certain malignant cells, inculture, demonstrate a greater or absolute reliance on an autocrinegrowth mechanism (3). Malignant cells which observe this autocrinebehavior circumvent the regulation of growth factor production by othercells and are therefore unregulated in their growth.

[0005] Study of autocrine growth control advances understanding of cellgrowth mechanisms and leads to important advances in the diagnosis andtreatment of cancer. Toward this end, a number of growth factors havebeen studied, including insulin-like growth factors (“IGF1” and “IGF2”),gastrin-releasing peptide (“GRP”), transforming growth factors alpha andbeta (“TGF-a” and “TGF-b”), and epidermal growth factor (“EGF”).

[0006] The present invention is directed to a recently discovered growthfactor. This growth factor was first discovered in the culture medium ofhighly tumorigenic “PC cells,” an insulin-independent variant isolatedfrom the teratoma derived adipogenic cell line 1246. This growth factoris referred to herein as “GP88.” GP88 has been purified and structurallycharacterized (4). Amino acid sequencing of GP88 indicates that GP88 hasamino acid sequence similarities with the mouse granulin/epithelinprecursor.

[0007] Granulins/epithelins (“grn/epi”) are 6 kDa polypeptides andbelong to a novel family of double cysteine rich polypeptides (5, 6).U.S. Pat. No. 5,416,192 (Shoyab et al.) is directed to 6 kDa epithelins,particularly epithelin 1 and epithelin 2. According to Shoyab, bothepithelins are encoded by a common 63.5 kDa precursor, which isprocessed into smaller forms as soon as it is synthesized, so that theonly natural products found in biological samples are the 6 kDa forms.Shoyab et al. teaches that the epithelin precursor is biologicallyinactive.

[0008] Contrary to the teachings of Shoyab et al., the inventor'slaboratory has demonstrated that the precursor is not always processedas soon as it is synthesized. Studies, conducted in part by thisinventor, have demonstrated that the precursor (i.e., GP88) is in factsecreted as an 88 kDa glycoprotein with an N-linked carbohydrate moietyof 20 kDa (4). Analysis of the N-terminal sequence of GP88 indicatesthat GP88 starts at amino acid 17 of the grn/epi precursor,demonstrating that the first 17 amino acids from the protein sequencededuced from the precursor cDNA correspond to a signal peptidecompatible with targeting for membrane localization or for secretion(4).

[0009] Also in contrast to the teachings of Shoyab et al., GP88 isbiologically active and has growth promoting activity, particularly asan autocrine growth factor for the producer cells.

SUMMARY OF INVENTION

[0010] The inventor has now unexpectedly discovered that a glycoprotein(GP88), which is expressed in a tightly regulated fashion in normalcells, is overexpressed and unregulated in highly tumorigenic cellsderived from the normal cells, that GP88 acts as a stringently requiredgrowth stimulator for the tumorigenic cells and that inhibition of GP88expression or action in the tumorigenic cells results in an inhibitionof the tumorigenic properties of the overproducing cells.

[0011] It is an object of this invention to provide GP88 antagonizingcompositions capable of inhibiting the expression or activity of GP88.

[0012] A further object of the invention is to provide methods fortreating diseases associated with a defect in GP88 quantity or activitysuch as but not limited to cancer im a mammal.

[0013] Another object of the invention is to provide methods fordetermining the susceptibility of a subject to diseases associated witha defect in GP88 expression or action.

[0014] Yet another object of the invention is to provide methods formeasuring susceptibility to GP88 antagonizing therapy.

[0015] Yet another object of the invention is to provide methods,reagents, and kits for the in vitro and in vivo detection of GP88 andtumorigenic activity in cells.

[0016] Additional objects and advantages of the invention will be setforth in part in the description that follows, and in part will beobvious from the description, or may be learned by the practice of theinvention.

[0017] To achieve the objects and in accordance with the purpose of theinvention, as embodied and properly described herein, the presentinvention provides compositions for diagnosis and-treatment of diseasessuch as but not limited to cancer in which cells exhibit an alteredexpression of GP88 or altered response to GP88.

[0018] Use of the term “altered expression” herein means increasedexpression or overexpression of GP88 by a factor of at least two-fold,and at times by a factor of 10 or more, based on the level of mRNA orprotein as compared to corresponding normal cells or surroundingperipheral cells. The term “altered expression” also means expressionwhich became unregulated or constitutive without being necessarilyelevated. Use of the terms increased or altered “response” to GP88 meansa condition wherein increase in any of the biological functions (e.g.,growth, differentiation, viral infectivity) conferred by GP88 results inthe same or equivalent condition as altered expression of GP88.

[0019] Use of the term “GP88” herein means epithelin/granulin precursorin cell extracts and extracellular fluids, and is intended to includenot only GP88 according to the amino acid sequences included in FIGS. 8or 9, which are of mouse and human origins, but also GP88 of otherspecies. In addition, the term also includes functional derivativesthereof having additional components such as a carbohydrate moietyincluding a glycoprotein or other modified structures.

[0020] Also intended by the term GP88 is any polypeptide fragment havingat least 10 amino-acids present in the above mentioned sequences.Sequences of this length are useful as antigens and for makingimmunogenic conjugates with carriers for the production of antibodiesspecific for various epitopes of the entire protein. Such polypeptidesare useful in screening such antibodies and in the methods directed todetection of GP88 in biological fluids. It is well known in the art thatpeptides are useful in generation of antibodies to larger proteins (7).In one embodiment of this invention, it is shown that peptides from12-19 amino-acids in length have been successfully used to developantibodies that recognize the full length GP88.

[0021] The polypeptide of this invention may exist covalently ornon-covalently bound to another molecule. For example, it may be fusedto one or more other polypeptides via one or more peptide bonds such asglutathione transferase, poly-histidine, or myc tag.

[0022] The polypeptide is sufficiently large to comprise anantigenetically distinct determinant or epitope which can be used as animmunogen to reproduce or test antibodies against GP88 or a functionalderivative thereof.

[0023] One embodiment includes the polypeptide substantially free ofother mammalian peptides. GP88 of the present invention can bebiochemically or immunochemically purified from cells, tissues or abiological fluid. Alternatively, the polypeptide can be produced byrecombinant means in a prokaryotic or eukaryotic expression system andhost cells.

[0024] “Substantially free of other mammalian polypeptides” reflects thefact that the polypeptide can be synthesized in a prokaryotic or anon-mammalian or mammalian eukaryotic organism, if desired.Alternatively, methods are well known for the synthesis of polypeptidesof desired sequences by chemical synthesis on solid phase supports andtheir subsequent separation from the support. Alternatively, the proteincan be purified from tissues or fluids of mammals where it naturallyoccurs so that it is at least 90% pure (on a weight basis) or even 99%pure, if desired, of other mammalian polypeptides, and is thereforesubstantially free from them. This can be achieved by subjecting thetissue extracts or fluids to standard protein purification such as onimmunoabsorbants bearing antibodies reactive against the protein. Oneembodiment of the present invention describes purification methods forthe purification of naturally occurring GP88 and of recombinant GP88expressed in baculovirus infected insect cells. Alternatively,purification from such tissues or fluids can be achieved by acombination of standard methods such as but not limited to the onesdescribed in reference (4).

[0025] As an alternative to a native purified or recombinantglycoprotein or polypeptide, “GP88” is intended to also includefunctional derivatives. By functional derivative is meant a “fragment,”“variant,” “analog,” or “chemical derivative” of the protein orglycoprotein as defined below. A functional derivative retains at leasta portion of the function of the full length GP88 which permits itsutility in accordance with the present invention.

[0026] A “fragment” of GP88 refers to any subset of the molecule that isa shorter peptide. This corresponds for example but is not limited toregions such as K19T and S14R for mouse GP88, and E19V and A14R(equivalent to murine K19T and S14R, respectively) for human GP88.

[0027] A “variant” of GP88 refers to a molecule substantially similar toeither the entire peptide or a fragment thereof. Variant peptides may beprepared by direct chemical synthesis of the variant peptide usingmethods known in the art.

[0028] Alternatively, amino acid sequence variants of the peptide can beprepared by modifying the DNA which encodes the synthesized protein orpeptide. Such variants include, for example, deletions, insertions, orsubstitutions of residues within the amino-acid sequence of GP88. Anycombination of deletion, insertion, and substitution may also be made toarrive at the final construct, provided the final construct possessesthe desired activity. The mutation that will be made in the DNA encodingthe variant peptide must not alter the reading frame and preferably willnot create complementary regions that could produce secondary mRNAstructures. At the genetic level these variants are prepared by sitedirected mutagenesis (8) of nucleotides in the DNA encoding the peptidemolecule thereby producing DNA encoding the variant, and thereafterexpressing the DNA in recombinant cell culture. The variant typicallyexhibits the same qualitative biological activity as the nonvariantpeptide.

[0029] An “analog” of GP88 protein refers to a non-natural moleculesubstantially similar to either the entire molecule or a fragmentthereof.

[0030] A “chemical derivative” contains additional chemical moieties notnormally a part of the peptide or protein. Covalent modifications of thepeptide are also included within the scope of this invention. Suchmodifications may be introduced into the molecule by reacting targetedamino-acid residues of the peptide with an organic derivatizing agentthat is capable of reacting with selected side chains or terminalamino-acid residues. Most commonly derivatized residues are cysteinyl,histidyl, lysinyl, arginyl, tyrosyl, glutaminyl, asparaginyl and aminoterminal residues. Hydroxylation of proline and lysine, phosphorylationof hydroxyl groups of seryl and threonyl residues, methylation of thealpha-amino groups of lysine, histidine, and histidine side chains,acetylation of the N-terminal amine and amidation of the C-terminalcarboxylic groups. Such derivatized moieties may improve the solubility,absorption, biological half life and the like. The moieties may alsoeliminate or attenuate any undesirable side effect of the protein andthe like. In addition, derivatization with bifunctional agents is usefulfor cross-linking the peptide to water insoluble support matrices or toother macromolecular carriers. Commonly used cross-linking agentsinclude glutaraldehyde, N-hydroxysuccinimide esters, homobifunctionalimidoesters, 1,1-bis(-diazoloacetyl)-2-phenylethane, and bifunctionalmaleimides. Derivatizing agents such asmethyl-3-[9p-azidophenyl)]dithiopropioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287 and 3,691,016 may be employed for proteinimmobilization.

[0031] Use of the term GP88 “antagonizing agents” herein means anycomposition that inhibits or blocks GP88 expression, production orsecretion, or any composition that inhibits or blocks the biologicalactivity of GP88. This can be achieved by any mode of action such as butnot limited to the following:

[0032] (A) GP88 antagonizing agents include any reagent or moleculeinhibiting GP88 expression or production including but not limited to:

[0033] (1) antisense GP88 DNA or RNA molecules that inhibit GP88expression by inhibiting GP88 translation;

[0034] (2) reagents (hormones, growth factors, small molecules) thatinhibit GP88 mRNA and/or protein expression at the transcriptional,translational or post-transaltional levels;

[0035] (3) factors, reagents or hormones that inhibit GP88 secretion;

[0036] (B) GP88 antagonizing agents also include any reagent or moleculethat will inhibit GP88 action or biological activity such as but notlimited to:

[0037] (1) neutralizing antibodies to GP88 that bind the protein andprevent it from exerting its biological activity;

[0038] (2) antibodies to the GP88 receptor that prevent GP88 frombinding to its receptor and from exerting its biological activity;

[0039] (3) competitive inhibitors of GP88 binding to its receptors;

[0040] (4) inhibitors of GP88 signaling pathways.

[0041] Specific examples presented herein provide a description ofpreferred embodiments, particularly the use of neutralizing antibodiesto inhibit GP88 biological action and the growth of the highlytumorigenic PC cells; the use of antisense GP88 cDNA and antisense GP88oligonucleotides to inhibit GP88 expression leading to inhibition of thetumorigenic properties of the PC cells; characterization of GP88receptors on cell surfaces of several cell lines including the mammaryepithelial cell line C57MG, the 1246 and PC cell lines and the mink lungepithelial cell line CCL64.

[0042] In one embodiment of the invention, the GP88 antagonizing agentsare antisense oligonucleotides to GP88. The antisense oligonucleotidespreferably inhibit GP88 expression by inhibiting translation of the GP88protein.

[0043] Alternatively, such a composition may comprise reagents, factorsor hormones that inhibit GP88 expression by regulating GP88 genetranscriptional activity. Such a composition may comprise reagents,factors or hormones that inhibit GP88 post-translational modificationand its secretion. Such a composition may comprise reagents that act asGP88 antagonists that block GP88 activity by competing with GP88 forbinding to GP88 cell surface receptors. Alternatively, such acomposition may comprise factors or reagents that inhibit the signalingpathway transduced by GP88 once binding to its receptors on diseasedcells.

[0044] Alternatively, the composition may comprise reagents that blockGP88 action such as an antibody specific to GP88 that neutralizes itsbiological activity, or an antibody to the GP88 receptor that blocks itsactivity.

[0045] The antibodies of the invention (neutralizing and others) arepreferably used as a treatment for cancer or other diseases in cellswhich exhibit an increased expression of GP88. By the term“neutralizing” it shall be understood that the antibody has the abilityto inhibit or block the normal biological activity of GP88, includingGP88's ability to stimulate cell proliferation or to induce tumor growthin experimental animals and in humans. An effective amount of anti-GP88antibody is administered to an animal, including humans, by variousroutes. In an alternative embodiment, the anti-GP88 antibody is used asa diagnostic to detect cells which exhibit an altered (increased)expression of GP88 as occurring in diseases such as but not limited tocancers, and to identify diseased cells whose growth is dependent onGP88 and which will respond to GP88 antagonizing therapy. In yet anotherembodiment, the anti-GP88 antibody is used to deliver compounds such ascytotoxic factors or antisense oligonucleotides to cells expressing orresponsive to GP88.

[0046] The antisense oligonucleotides of the invention are also used asa treatment for cancer in cells which exhibit an increased expression ofGP88. An effective amount of the antisense oligonucleotide isadministered to an animal, including humans, by various routes.

[0047] The present invention also provides a method for determining thesusceptibility to diseases associated with a defect in GP88 expressionor action which comprises obtaining a sample of biological fluid ortissue and measuring the amount of GP88 in the fluid or tissue ormeasuring the susceptibility of the cells to respond to GP88. In thecase of cancer, the amount of GP88 being proportional to thesusceptibility to the cancer.

[0048] The present invention also provides a method for measuring thedegree of severity of cancer which comprises obtaining a sample ofbiological fluid or tissue and measuring the amount of GP88 in the fluidor tissue sample, the amount of GP88 being proportional to the degree orseverity of the cancer.

[0049] The present invention also provides a method for measuringsusceptibility to GP88 antagonizing therapy which comprises obtaining asample of the diseased tissue (biopsy), maintaining the cells derivedfrom the sample in culture, treating the cells derived from the culturewith anti-GP88 neutralizing antibody and determining if the neutralizingantibody inhibits the cell growth. Ability of the antibody to inhibitcell growth is indicative that the cells are dependent on GP88 toproliferate and is predictive that GP88 antagonizing therapy will beefficacious.

[0050] The present invention also provides a method for determining thesusceptibility to cancer associated with an abnormality in GP88 receptorlevel or activity which comprises obtaining a sample of tissue andmeasuring the amount of GP88 receptor protein or mRNA in the tissue ormeasuring the tyrosine kinase activity of the receptor in the tissue(GP88 binding to its receptor induces tyrosine phosphorylation ofcellular proteins including the receptor for G88).

[0051] The present invention also provides a method for targeting GP88antagonizing reagents to the diseased site by conjugating them to ananti-GP88 antibody or an anti-GP88 receptor antibody.

[0052] The accompanying drawings, which are incorporated in andconstitute a part of this specification, illustrate embodiments of theinvention and, together with the description, serve to explain theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0053]FIG. 1A compares the level of expression of GP88 protein in the1246, 1246-3A and PC cell lines. Cells were cultured in DME-F12 mediumsupplemented with 2% fetal bovine serum (FBS). GP88 expression levelswere measured by immunoprecipitation and Western blot analysis withanti-K19T antibody.

[0054]FIG. 1B compares the level of GP88 mRNA expression in the 1246,1246-3A and PC cell lines. mRNA for RPL32 is used as an internal controlfor equal amounts of RNA loading.

[0055]FIG. 1C compares the expression of GP88 mRNA in 1246 cells (leftpanel) and in PC cells (right panel) in serum-free and serum containingmedium. The results show that GP88 expression in 1246 cells is inhibitedby the addition of fetal bovine serum whereas such inhibition is notobserved in the highly tumorigenic PC cells.

[0056]FIG. 2 illustrates the effect of treatment of the highlytumorigenic PC cells with increasing concentrations of anti-GP88neutralizing antibody.

[0057]FIG. 3 shows C3H mice injected subcutaneously with 10⁶ antisenseGP88 transfected PC cells (bottom) and with empty vector transfectedcontrol PC cells (top).

[0058]FIG. 4 shows in vivo GP88 expression levels in C3H mice tumortissues and in surrounding normal tissues.

[0059]FIG. 5 shows GP88 mRNA expression levels in estrogen receptorpositive and estrogen receptor negative human mammary carcinoma celllines.

[0060]FIG. 6 shows the effect of increasing concentrations of GP88 onthe growth of the mouse mammary epithelial cell line C57.

[0061]FIG. 7 shows the growth properties and tumorigenic ability of PCcells transfected with a cytomegalovirus promoter controlled expressionvector containing GP88 in antisense orientation and PC cells transfectedwith an empty vector.

[0062]FIG. 8 shows the nucleotide and deduced amino-acid sequence ofmouse GP88. Peptide regions used as antigens to raise anti-GP88antibodies K(19T and S14R are underlined. The region cloned in theantisense orientation in the pCMV4 mammalian expression vector isindicated between brackets.

[0063]FIG. 9A shows the nucleotide sequence of human GP88 cDNA.Indicated between brackets is the region cloned in the antisenseorientation into the pcDNA3 mammalian expression system; and

[0064]FIG. 9B shows the deduced amino-acid sequence of human GP88. TheE19V region used as antigen to develop anti-human GP88 neutralizingantibody is underlined. It also indicates the region A14R equivalent tothe mouse S14R region.

[0065]FIG. 10 shows the amino-acid sequence of mouse GP88 arranged toshow the 7 and one-half repeats defined as granulins g, f, B, A, C, Dand e (right side). This representation shows that the region K19T andS14R used to raise GP88 antibodies for developing anti-GP88 neutralizingantibodies is found between two epithlin/granulin repeats in what isconsidered a variant region. Indicated on the right hand side is thegranulin classification of the repeats according to Bateman et al (6).Granulin B and granulin A are also defined as epithelin 2 and epithelin1 respectively according to Plowman et al., 1992 (5).

[0066]FIG. 11 shows a schematic representation of pCMV4 and a GP88 cDNAclone indicating the restriction sites used to clone GP88 antisense cDNAinto the expression vector.

[0067]FIG. 12 shows the cross-linking of ¹²⁵I-rGP88 to GP88 cell surfacereceptors on CCL-64 cells. The cross-linking reaction was carried outwith disuccinimidyl suberate (DSS). Reaction products were analyzed bySDS-PAGE on a 7% polyacrylamide gel.

[0068]FIG. 13 shows the cross-lining of ¹²⁵I-rGP88 to GP88 cell surfacereceptors on 3T3 fibroblasts, PC cells and C57MG mammary epithelialcells. The results show that these various cell lines display GP88 cellsurface receptors of similar molecular weight as the ones on CCL64 cells(FIG. 12).

[0069]FIG. 14 shows GP88 expression levels in non-tumorigenic MCF 10Aand in malignant (MCF 7, MDA-468) human mammary epithelial cells.

[0070]FIG. 15 shows that GP88 expression is inhibited by antisense GP88cDNA transfection in human breast carcinoma MDA-468 cells.

DESCRIPTION OF THE INVENTION

[0071] Reference will now be made in detail to the presently preferredembodiments of the invention, which, together with the followingexamples, serve to explain the principles of the invention.

Biological Activity of GP88

[0072] The invention relates to GP88 and antitumor and antiviralcompositions useful for treating and diagnosing diseases linked toaltered (increased) expression of GP88. Alternatively this invention isused for treating and diagnosing diseases linked to increasedresponsiveness to GP88. Using a murine model system consisting of threecell lines, the inventor has shown that cells which overexpress GP88form tumors. The parent cell line, 1246, is a C3H mouse adipogenic cellline which proliferates and differentiates into adipocytes in a definedmedium under stringent regulation by insulin (9, 10). The 1246 cellscannot form tumors in a syngeneic animal (C3H mouse) even when injectedat a high cell density. An insulin independent cell line, 1246-3A, wasisolated from 1246 cells maintained in insulin-free medium (11). The1246-3A cells lost the ability to differentiate and form tumors when 10⁶are injected subcutaneously in syngeneic mice. A highly tumorigenic cellline, PC, was developed from 1246-3A cells by an in vitro-in vivoshuttle technique. The PC cells formed tumors when 10⁴ cells wereinjected into syngeneic mice (12).

[0073] GP88 is overexpressed in the insulin-independent tumorigenic celllines relative to the parent non-tumorigenic insulin-dependent cellline. Moreover, the degree of overexpression of GP88 positivelycorrelates with the degree of tumorigenicity of these cells,demonstrating for the first time that GP88 is important in tumorigenesis(FIG. 1). With reference to FIG. 1, since GP88 is synthesized by cellsbut also secreted in culture medium, the level of GP88 was determined incell lysates and in culture medium (CM). All cells were cultivated inDME/F12 nutrient medium supplemented with 2% fetal bovine serum. Whencells reached confluency, culture medium (CM) was collected and celllysates were prepared by incubation in buffer containing detergentfollowed by a 10,000×g centrifugation. Cell lysate and conditionedmedium were normalized by cell number. Samples from cell lysate andconditioned medium were analyzed by Western blot analysis using ananti-GP88 antibody, as explained below.

[0074] The development of a neutralizing antibody confirmed GP88's keyrole in tumorigenesis. When an anti-GP88 antibody directed to the K19Tregion of mouse GP88 was added to the culture medium, the growth ofhighly tumorigenic PC cells was inhibited in a dose dependent fashion(FIG. 2). With reference to FIG. 2, PC cells were cultivated in 96 wellplates at a density 2×10⁴ cells/well in DME/F12 medium supplemented withhuman fibronectin (2 μg/ml) and human transferrin (10 μg/ml). Increasingconcentrations of anti-GP88 IgG fraction were added to the wells afterthe cells were attached. Control cells were treated with equivalentconcentrations of non-immune IgG. Two days later, 0.25 mCi of³H-thymidine was added per well for 6 hrs. Cells were then harvested tocount ³H-thymidine incorporated into DNA as a measure for cellproliferation.

[0075] Moreover, when the expression of GP88 was specifically inhibitedby antisense GP88 cDNA in PC cells, the production of GP88 was reducedand these PC cells could no longer form tumors in syngeneic C3H mouse.In addition, these PC cells regained responsiveness to insulin. Withreference to FIG. 3 and Tables 1 and 2, C3H female mice were injectedsubcutaneously with 10⁶ antisense GP88 transfected PC cells (asexplained below) or 10⁶ empty vector transfected PC cells. Mice weremonitored daily for tumor appearance. Photographs were taken 45 daysafter injection of the cells. The results show that mice injected withantisense GP88 PC cells do not develop tumors, in contrast to the miceinjected with empty vector transfected PC cells used as control. TABLE 1COMPARISON OF TUMORIGENIC PROPERTIES OF GP88 ANTISENSE TRANSFECTEDCELLS, CONTROL TRANSFECTED CELLS AND PC CELLS AVERAGE DAY OF NUMBER OFAVERAGE CELLS TUMOR MICE WITH TUMOR INJECTED DETECTION TUMORS WEIGHT (g)PC 15 ± 3.0 5/5 9.0 ± 3.2 P14 15 ± 3.7 5/5 7.8 ± 2.7 ASGP88 — 0/5 —

[0076] Tumors were excised and weighed at 45 days. —indicates no tumorformation. TABLE 2 COMPARISON OF PROPERTIES OF 1246, PC CELLS AND GP88ANTISENSE CELLS insulin GP88 antisense independence transfection 1246cells PC cells Antisense GP 88 cells insulin responsiveinsulin-independent for recovery of insulin for growth and growthdifferentiation responsiveness for differentiation deficient growth(differentiation?) autocrine production of insulin-related factor cellsurface insulin cell surface insulin receptor cell surface insulinreceptor expression expression very low receptor expression highelevated GP88 expression GP88 expression GP88 expression lowconstitutively high inhibited by antisense GP88 expression No inhibitionby serum inhibited by serum GP88 expression GP88 expression recovery ofinsulin regulated by insulin constitutive regulation for endogenous GP88expression non-tumorigenic highly tumorigenic non-tumorigenic

[0077] Comparison of the expression of GP88 indicates that in vivo GP88levels in tumors is dramatically higher than in normal tissues (FIG. 4).C3H mice were injected with 10⁶ PC cells. Tumor bearing mice wereeuthanized. Tumors, fat pads and connective tissue were collected. Celllysates were prepared by incubation in buffer containing detergent asdescribed above for FIG. 1. Protein concentration of tissue extracts wasdetermined, and equivalent amounts of proteins for each sample wereanalyzed by SDS-PAGE followed by Western blot analysis usinganti-GP88-antibody to measure the content of GP88 in tissue extracts.The results showed that the level of GP88 in tumor extracts is at least10-fold higher than in surrounding connective and fat tissues.

[0078] In normal cells (1246 cells, fibroblasts), the expression of GP88is regulated, in particular by insulin, and inhibited by fetal bovineserum. In tumorigenic cells, a loss of regulation of normal growth leadsto the increased expression of GP88 and the acquisition of GP88dependence for growth. Therefore, inhibition of GP88 expression and/oraction is an effective approach to suppression of tumorigenesis.Detection of an elevated GP88 expression in biopsies provides diagnosticanalysis of tumors that are responsive to GP88 inhibition therapy.

[0079] GP88 is also a tumor inducing factor in human cancers. As seen inthe 1246-3A cell line, a loss of responsiveness to insulin (or to IGF-I)and a concurrent increase in malignancy has been well documented (13,14) in several human cancers including but not limited to breastcancers. Specifically, breast carcinoma is accompanied by theacquisition of an insulin/IGF-I autocrine loop, which is also thestarting point of the development of tumorigenic properties in the mousemodel system discussed above. Furthermore, GP88 expression is elevatedin human breast carcinomas. More specifically, with reference to FIG. 5,human GP88 was highly expressed in estrogen receptor positive and alsoin estrogen receptor negative insulin/IGF-I independent highly malignantcells. Also, GP88 is a potent growth factor for mammary epithelial cells(FIG. 6). The data in FIG. 5 was obtained by cultivating MCF7,MDA-MB-453 and MDA-MB-468 cells in DME/F12 medium supplemented with 10%fetal bovine serum (FBS). RNA was extracted from each cell line by theRNAzol method and poly-A⁺ RNA prepared. GP88 mRNA expression wasexamined by Northern blot analysis with 3 μg of poly-A⁺ RNA for eachcell line using a ³²P-labeled GP88 cDNA probe.

[0080] For Northern blot analysis of GP88 mRNA expression in rodentcells or tissues (mouse and rats), we used a mouse GP88 cDNA probe 311bp in length starting at nucleotide 551 to 862 (corresponding toamino-acid sequence 160 to 270). RNA can be extracted by a variety ofmethods (Sambrook, Molecular Biology manual: 35) well known to people ofordinary skill in the art. The method of choice was to extract RNA usingRNAzol (Cinnabiotech) or Trizol (Gibco-BRL) solutions which consists ofa single step extraction by guanidinium isothiocyanate and phenol-chloroform.

[0081] For Northern blot analysis of GP88 mRNA expression in human celllines, a 672 bp human GP88 cDNA probe was developed corresponding tonucleotide 1002 to 1674 (corresponding to amino-acid sequence 334-558)of human GP88. See example 8 for a detailed and specific description ofthe Northern blot analysis method used in the preferred embodiments.

[0082] With respect to FIG. 6, C57MG cells were cultivated in thepresence of increasing concentrations of GP88 purified from PC cellsconditioned medium (top panel), and recombinant GP88 expressed in insectcells (bottom panel), to demonstrate the growth stimulating effect ofincreasing concentrations of GP88 on the growth of the mouse mammaryepithelial cell line C57MG.

[0083] A correlation between IGF-I autocrine production and increasedmalignancy has also been well established for glioblastomas,teratocarcinomas and breast carcinomas (2, 15, 16, 17). In thesecancers, GP88 expression is also elevated in human tumors when comparedto non-tumorigenic human fibroblasts and other human cell lines. GP88promotes the growth of mammary carcinoma cells.

Anti-GP88 Antibodies

[0084] The invention provides compositions for treating and diagnosingdiseases linked to increased expression of GP88. This also will apply totreatment and diagnosis of diseases linked to increased responsivenessto GP88. The compositions of this invention include anti-GP88 antibodieswhich neutralize the biological activity of GP88.

[0085] The present invention is also directed to an antibody specificfor an epitope of GP88 and the use of such antibody to detect thepresence or measure the quantity or concentration of GP88 molecule, afunctional derivative thereof or a homologue from different animalspecies in a cell, a cell or tissue extract, culture medium orbiological fluid. Moreover, antibody can be used to target cytotoxicmolecules to a specific site.

[0086] For use as antigen for development of antibodies, the GP88protein naturally produced or expressed in recombinant form orfunctional derivative thereof, preferably having at least 9 amino-acids,is obtained and used to immunize an animal for production of polyclonalor monoclonal antibody. An antibody is said to be capable of binding amolecule if it is capable of reacting with the molecule to thereby bindthe molecule to the antibody. The specific reaction is meant to indicatethat the antigen will react in a highly selective manner with itscorresponding antibody and not with the multitude of other antibodieswhich may be evoked by other antigens.

[0087] The term antibody herein includes but is not limited to human andnon-human polyclonal antibodies, human and non-human monoclonalantibodies (mAbs), chimeric antibodies, anti-idiotypic antibodies(anti-IdAb) and humanized antibodies. Polyclonal antibodies areheterogeneous populations of antibody molecules derived either from seraof animals immunized with an antigen or from chicken eggs. Monoclonalantibodies (“mAbs”) are substantially homogeneous populations ofantibodies to specific antigens. mAbs may be obtained by methods knownto those skilled in the art (18, 19, 20 and U.S. Pat. No. 4,376,110).Such antibodies may be of any immunological class including IgG, IgM,IgE, IgA, IgD and any subclass thereof The hybridoma producing human andnon-human antibodies to GP88 may be cultivated in vitro or in vivo. Forproduction of a large amount of mAbs, in vivo is the presently preferredmethod of production. Briefly, cells from the individual hybridomas areinjected intraperitoneally into pristane primed Balb/c mice or Nude miceto produce ascites fluid containing high concentrations of the desiredmAbs. mAbs may be purified from such ascites fluids or from culturesupernatants using standard chromatography methods well known to thoseof skill in the art.

[0088] Human monoclonal Ab to human GP88 can be prepared by immunizingtransgenic mice expressing human immunoglobulin genes. Hybridomaproduced by using lymphocytes from these transgenic animals will producehuman immunoglobulin instead of mouse immunoglobulin.

[0089] Since most monoclonal antibodies are derived from murine sourceand other non-human sources, their clinical efficiency may be limiteddue to the immunogenicity of rodent mAbs administered to humans, weakrecruitment of effector function and rapid clearance from serum (25). Tocircumvent these problems, the antigen-binding properties of murineantibodies can be conferred to human antibodies through a process calledhumanization (25). A humanized antibody contains the amino-acidsequences for the 6 complementarity-determining regions (CDRs) of theparent murine mAb which are grafted onto a human antibody framework. Thelow content of non-human sequences in humanized antibodies (around 5%)has proven effective in both reducing the immunogenicity and prolongingthe serum half life in humans. Methods such as the ones using monovalentphage display and combinatorial library strategy (26, 27) forhumanization of monoclonal antibodies are now widely applied to thehumanization of a variety of antibodies and are known to people skilledin the art. These humanized antibodies and human antibodies developedwith transgenic animals as described above are of great therapeutic usefor several diseases including but not limited to cancer.

[0090] Hybridoma supernatants and sera are screened for the presence ofantibody specific for GP88 by any number of immunoassays including dotblots and standard immunoassays (EIA or ELISA) which are well known inthe art. Once a supernatant has been identified as having an antibody ofinterest, it may be further screened by Western blotting to identify thesize of the antigen to which the antibody binds. One of ordinary skillin the art will know how to prepare and screen such hybridomas withoutundue experimentation in order to obtain a desired polyclonal or mAb.

[0091] Chimeric antibodies have different portions derived fromdifferent animal species. For example, a chimeric antibody might have avariable region from a murine mAb and a human immunoglobulin constantregion. Chimeric antibodies and methods for their production are alsoknown to those skilled in the art (21-24).

[0092] An anti-idiotypic (“anti-IdAb”) is an antibody which recognizesunique determinants generally associated with the antigen-binding siteof an antibody. An anti-IdAb can be prepared by immunizing an animal ofthe same species and genetic type (e.g., mouse strain) as the source ofthe mAb with the mAb to which an anti-IdAb is being prepared. Theimmunized animal will recognize and respond to the idiotypicdeterminants of the immunizing antibody by producing antibody to theseidiotypic determinants (the anti-IdAb). The anti-IdAb may also be usedas an immunogen to produce an immune response in yet another animal,producing a so-called anti-anti-IdAb. The anti-anti-IdAb may beepitopically identical to the original mAb which induced the anti-IdAb.Thus by using antibodies to the idiotypic determinants of a mAb, it ispossible to identify other clones expressing antibodies of identicalspecificity.

[0093] Accordingly, mAbs generated against GP88 may be used to inducehuman and non-human anti-IdAbs in suitable animals. Spleen cells fromsuch immunized mice are used to produce hybridomas secreting human ornon-human anti-Id mAbs. Further, the anti-Id mAbs can be coupled to acarrier such as Keyhole Limpet Hemocyanin (KLH) or bovine serum albumin(BSA) and used to immunize additional mice. Sera from these mice willcontain human or non-human anti-anti-IdAb that have the bindingproperties of the original mAb specific for a GP88 polypeptide epitope.The anti-Id mAbs thus have their own idiotypic epitopes or idiotypesstructurally similar to the epitope being evaluated.

[0094] The term antibody is also meant to include both intact moleculesas well as fragments thereof such as, for example, Fab and F(ab′)2,which are capable of binding to the antigen. Fab and F(ab′)2 fragmentslack the Fc fragment of intact antibody, clear more rapidly from thecirculation and may have less non-specific tissue binding than an intactantibody (28). Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to generate Fab fragments) andpepsin (to generate F(ab′)2 fragments). It will be appreciated that Faband F(ab′)2 and other fragments of the antibodies useful in the presentinvention may be used for the detection or quantitation of GP88, and fortreatment of pathological states related to GP88 expression, accordingto the methods disclosed herein for intact antibody molecules.

[0095] According to the present invention, antibodies that neutralizeGP88 activity in vitro can be used to neutralize GP88 activity in vivoto treat diseases associated with increased GP88 expression or increasedresponsiveness to GP88, such as but not limited to cancer and viralinfection. A subject, preferably a human subject, suffering from diseaseassociated with increased GP88 expression is treated with an antibody toGP88. Such treatment may be performed in conjunction with otheranti-cancer or anti-viral therapy. A typical regimen comprisesadministration of an effective amount of the antibody specific for GP88administered over a period of one or several weeks and including betweenabout one and six months. The antibody of the present invention may beadministered by any means that achieves its intended purpose. Forexample, administration may be by various routes including but notlimited to subcutaneous, intravenous, intradermal, intramuscular,intraperitoneal and oral. Parenteral administration can be by bolusinjection or by gradual perfusion over time. Preparations for parenteraladministration include sterile aqueous or non-aqueous solutions,suspensions and emulsions, which may contain auxiliary agents orexcipients known in the art. Pharmaceutical compositions such as tabletsand capsules can also be prepared according to routine methods. It isunderstood that the dosage of will be dependent upon the age, sex andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment and the nature of the effect desired. The ranges ofeffective doses provided below are not intended to limit the inventionand merely represent preferred dose ranges. However the most preferreddosage will be tailored to the individual subject as is understood anddeterminable by one skilled in the art. The total dose required for eachtreatment may be administered by multiple doses or in a single dose.Effective amounts of antibody are from about 0.01 μg to about 100 mg/kgbody weight and preferably from about 10 μg to about 50 mg/kg. Antibodymay be administered alone or in conjunction with other therapeuticsdirected to the same disease.

[0096] According to the present invention and concerning theneutralizing antibody, GP88 neutralizing antibodies can be used in alltherapeutic cases where it is necessary to inhibit GP88 biologicalactivity, even though there may not necessarily be a change in GP88expression, including cases where there is an overexpression of GP88cell surface receptors and this in turn results in an increasedbiological activity, or where there is an alteration in GP88 signalingpathways or receptors leading to the fact that the signaling pathwaysare always “turned on.” Neutralizing antibodies to growth factor and togrowth factor receptors have been successfully used to inhibit thegrowth of cells whose proliferation is dependent on this growth factor.This has been the case for IGF-I receptor in human breast carcinomacells (14) and bombesin for lung cancer (29). The antibody to GP88 canalso be used to deliver compounds such as, but not limited to, cytotoxicreagents such as toxins, oncotoxins, mitotoxins and immunotoxins, orantisense oligonucleotides, in order to specifically target them tocells expressing or responsive to GP88 (30).

[0097] One region that allows antigen to develop a neutralizing antibodyto GP88 is the 19 amino-acid region defined as IK19T in the mouse GP88,and E19V in the human GP88 which is not located within theepithelin/granulin 6 kDa repeats but between these repeats, specificallybetween granulin A (epithelin 1) and granulin C (5) in what isconsidered a variant region (see FIG. 10). Without wishing to be boundby theory, it is believed that the region important for the biologicalactivity of GP88 lies outside of the epithelin repeats.

[0098] The antibodies or fragments of antibodies useful in the presentinvention may also be used to quantitatively or qualitatively detect thepresence of cells which express the GP88 protein. This can beaccomplished by immunofluorescence techniques employing a fluorescentlylabeled antibody (see below) with fluorescent microscopic, flowcytometric, or fluorometric detection. The reaction of antibodies andpolypeptides of the present invention may be detected by immunoassaymethods well known in the art (20).

[0099] The antibodies of the present invention may be employedhistologically as in light microscopy, immunofluorescence orimmunoelectron microscopy, for in situ detection of the GP88 protein intissues samples or biopsies. In situ detection may be accomplished byremoving a histological specimen from a patient and applying theappropriately labeled antibody of the present invention. The antibody(or fragment) is preferably provided by applying or overlaying thelabeled antibody (or fragment) to the biological sample. Through the useof such a procedure, it is possible to determine not only the presenceof the GP88 protein but also its distribution in the examined tissue.Using the present invention, those of ordinary skill in the art willreadily perceive that any wide variety of histological methods (such asstaining procedures) can be modified in order to achieve such in situdetection.

[0100] Assays for GP88 typically comprise incubating a biological samplesuch as a biological fluid, a tissue extract, freshly harvested orcultured cells or their culture medium in the presence of a detectablylabeled antibody capable of identifying the GP88 protein and detectingthe antibody by any of a number of techniques well known in the art.

[0101] The biological sample may be treated with a solid phase supportor carrier such as nitrocellulose or other solid support capable ofimmobilizing cells or cell particles or soluble proteins. The supportmay then be washed followed by treatment with the detectably labeledanti-GP88 antibody. This is followed by wash of the support to removeunbound antibody. The amount of bound label on said support may then bedetected by conventional means. By solid phase support is intended anysupport capable of binding antigen or antibodies such as but not limitedto glass, polystyrene polypropylene, nylon, modified cellulose, orpolyacrylamide.

[0102] The binding activity of a given lot of antibody to the GP88protein may be determined according to well known methods. Those skilledin the art will be able to determine operative and optimal assayconditions for each determination by employing routine experimentation.

[0103] Detection of the GP88 protein or functional derivative thereofand of a specific antibody for the protein may be accomplished by avariety of immunoassays well known in the art such as enzyme linkedimmunoassays (EIA) or radioimmunoassays (RIA). Such assays are wellknown in the art and one of skill will readily know how to carry outsuch assays using the anti-GP88 antibodies and GP88 protein of thepresent invention.

[0104] Such immunoassays are useful to detect and quantitate GP88protein in serum or other biological fluid as well as in tissues, cells,cell extracts, or biopsies. In a preferred embodiment, the concentrationof GP88 is measured in a tissue specimen as a means for diagnosingcancer or other disease associated with increased expression of GP88.

[0105] The presence of certain types of cancers and the degree ofmalignancy are said to be “proportional” to an increase in the level ofthe GP88 protein. The term “proportional” as used herein is not intendedto be limited to a linear or constant relationship between the level ofprotein and the malignant properties of the cancer. The term“proportional” as used herein, is intended to indicate that an increasedlevel of GP88 protein is related to appearance, recurrence or display ofmalignant properties of a cancer or other disease associated withincreased expression of GP88 at ranges of concentration of the proteinthat can be readily determined by one skilled in the art.

[0106] Another embodiment of the invention relates to evaluating theefficacy of anti-cancer or anti-viral drug or agent by measuring theability of the drug or agent to inhibit the expression or production ofGP88. The antibodies of the present invention are useful in a method forevaluating anti-cancer or anti-viral drugs in that they can be employedto determine the amount of the GP88 protein in one of theabove-mentioned immunoassays. Alternatively, the amount of the GP88protein produced is measured by bioassay (cell proliferation assay) asdescribed herein. The bioassay and immunoassay can be used incombination for a more precise assessment.

[0107] An additional embodiment is directed to an assay for diagnosingcancers or other diseases associated with an increase in GP88 expressionbased on measuring in a tissue or biological fluid the amount of mRNAsequences present that encode GP88 or a functional derivative thereof,preferably using an RNA-DNA hybridization assay. The presence of certaincancers and the degree of malignancy is proportional to the amount ofsuch mRNA present. For such assays the source of mRNA will be biopsiesand surrounding tissues. The preferred technique for measuring theamount of mRNA is a hybridization assay using DNA of complementaritybase sequence.

[0108] Another related embodiment is directed to an assay for diagnosingcancers or other diseases associated with an increase in GP88responsiveness based on measuring on a tissue biopsy whether treatmentwith anti-GP88 neutralizing antibody will inhibit its growth or otherbiological activity.

[0109] Another related embodiment is a method for measuring the efficacyof anti-cancer or anti-viral drug or agent which comprises the steps ofmeasuring the agent's effect on inhibiting the expression of mRNA forGP88. Similarly such method can be used to identify or evaluate theefficacy of GP88 antagonizing agents by measuring the ability of saidagent to inhibit the production of GP88 mRNA.

[0110] Nucleic acid detection assays, especially hybridization assays,can be based on any characteristic of the nucleic acid molecule such asits size, sequence, or susceptibility to digestion by restrictionendonucleases. The sensitivity of such assays can be increased byaltering the manner in which detection is reported or signaled to theobserver. A wide variety of labels have been extensively developed andused by those of ordinary skill in the art, including enzymatic,radioisotopic, fluorescent, chemical labels and modified bases.

[0111] One method for overcoming the sensitivity limitation of a nucleicacid for detection is to selectively amplify the nucleic acid prior toperforming the assay. This method has been referred as the “polymerasechain reaction” or PCR (31 and U.S. Pat. Nos. 4,683,202 and 4,582,788).The PCR reaction provides a method for selectively increasing theconcentration of a particular nucleic acid sequence even when thatsequence has not been previously purified and is present only in asingle copy in a particular sample.

GP88 Antisense Components

[0112] This invention also provides GP88 antisense components. Theconstitutive expression of antisense RNA in cells has been shown toinhibit the expression of more than 20 genes and the list continues togrow (32-34). Possible mechanisms for antisense effects are the blockageof translation or prevention of splicing, both of which have beenobserved in vitro. Interference with splicing allows the use of intronsequences (30) which should be less conserved and therefore result ingreater specificity, inhibiting expression of a gene product of onespecies but not its homologue in another species.

[0113] The term antisense component corresponds to an RNA sequence aswell as a DNA sequence coding therefor, which is sufficientlycomplementary to a particular mRNA molecule, for which the antisense RNAis specific, to cause molecular hybridization between the antisense RNAand the mRNA such that translation of the mRNA is inhibited. Suchhybridization can occur under in vivo conditions. The action of theantisense RNA results in specific inhibition of gene expression in thecells (32-35).

[0114] According to the present invention, transfection of tumorigeniccells with DNA antisense to the GP88 cDNA inhibits endogenous GP88expression and inhibits tumorigenicity of the antisense cDNA transfectedcells. This antisense DNA must have sufficient complementarity, about18-30 nucleotides in length, to the GP88 gene so that the antisense RNAcan hybridize to the GP88 gene (or mRNA) and inhibit GP88 geneexpression regardless of whether the action is at the level of splicing,transcription, or translation. The degree of inhibition is readilydiscernible to one skilled in the art without undue experimentationgiven the teachings herein and preferably is sufficient to inhibit thegrowth of cells whose proliferation is dependent on the expression ofGP88. One of ordinary skill in the art will recognize that the antisenseRNA approach is but a number of known mechanisms which can be employedto block specific gene expression.

[0115] The antisense components of the present invention may behybridizable to any of several portions of the target GP88 cDNA,including the coding sequence, 3′ or 5′ untranslated regions, or otherintronic sequences, or to GP88 mRNA. As is readily discernible by one ofordinary skill in the art, the minimal amount of homology required bythe present invention is that sufficient to result in hybridization tothe GP88 DNA or mRNA and in inhibition of transcription of the DNA, ortranslation or function of the mRNA, preferably without affecting thefunction of other mRNA molecules and the expression of other unrelatedgenes.

[0116] Antisense RNA is delivered to a cell by transformation ortransfection via a vector, including retroviral vectors and plasmids,into which has been placed DNA encoding the antisense RNA with theappropriate regulatory sequences including a promoter to result inexpression of the antisense RNA in a host cell. Stable transfection ofvarious antisense expression vectors containing GP88 cDNA fragments inthe antisense orientation have been performed. One can also deliverantisense components to cells using a retroviral vector. Delivery canalso be achieved by liposomes.

[0117] For purpose of antisense technology for in vivo therapy, thecurrently preferred method is to use antisense oligonucleotides (32,36), instead of performing stable transfection of an antisense cDNAfragment constructed into an expression vector. Antisenseoligonucleotides having a size of 15-30 bases in length and withsequences hybridizable to any of several portions of the target GP88cDNA, including the coding sequence, 3′ or 5′ untranslated regions, orother intronic sequences, or to GP88 mRNA, are preferred. Sequences forthe antisense oligonucleotides to GP88 are preferably selected as beingthe ones that have the most potent antisense effects (37, 38). Factorsthat govern a target site for the antisense oligonucleotide sequence arerelated to the length of the oligonucleotide, binding affinity, andaccessibility of the target sequence. Sequences may be screened in vitrofor potency of their antisense activity by measuring inhibition of GP88protein translation and GP88 related phenotype, e.g., inhibition of cellproliferation in cells in culture. In general it is known that mostregions of the RNA (5′ and 3′ untranslated regions, AUG initiation,coding, splice junctions and introns) can be targeted using antisenseoligonucleotides.

[0118] The preferred GP88 antisense oligonucleotides are thoseoligonucleotides which are stable, have a high resilience to nucleases(enzymes that could potentially degrade oligonucleotides), possesssuitable pharmacokinetics to allow them to traffic to disease tissue atnon-toxic doses, and have the ability to cross through plasma membranes.

[0119] Phosphorothioate antisense oligonucleotides may be used (39).Modifications of the phosphodiester linkage as well as of theheterocycle or the sugar may provide an increase in efficiency. Withrespect to modification of the phosphodiester linkage, phophorothioatemay be used. An N3′-P5′ phosphoramidate linkage has been described asstabilizing oligonucleotides to nucleases and increasing the binding toRNA (40). Peptide nucleic acid (PNA) linkage is a complete replacementof the ribose and phosphodiester backbone and is stable to nucleases,increases the binding affinity to RNA, and does not allow cleavage byRNAse H. Its basic structure is also amenable to modifications that mayallow its optimization as an antisense component. With respect tomodifications of the heterocycle, certain heterocycle modifications haveproven to augment antisense effects without interfering with RNAse Hactivity. An example of such modification is C-5 thiazole modification.Finally, modification of the sugar may also be considered. 2′-O-propyland 2′-methoxyethoxy ribose modifications stabilize oligonucleotides tonucleases in cell culture and in vivo. Cell culture and in vivo tumorexperiments using these types of oligonucleotides targeted to c-raf-1resulted in enhanced potency. As general references for antisenseoligonucleotides, see (32-34)

[0120] The delivery route will be the one that provides the bestantisense effect as measured according to the criteria described above.In vitro cell culture assays and in vivo tumor growth assays usingantisense oligonucleotides have shown that delivery mediated by cationicliposomes, by retroviral vectors and direct delivery are efficient. (36,41-43) Another possible delivery mode is targeting using antibody tocell surface markers for the tumor cells. Antibody to GP88 or to itsreceptor may serve this purpose.

Recombinant GP88

[0121] The present invention is also directed to DNA expression systemsfor expressing a recombinant GP88 polypeptide or a functional derivativethereof substantially free of other mammalian DNA sequences. Such DNAmay be double or single stranded. The DNA sequence should preferablyhave about 20 or more nucleotides to allow hybridization to anotherpolynucleotide. In order to achieve higher specificity of hybridization,characterized by the absence of hybridization to sequences other thanthose encoding the GP88 protein or a homologue or functional derivativethereof, a length of at least 50 nucleotides is preferred.

[0122] The present invention is also directed to the above DNAmolecules, expressible vehicles or vectors as well as hosts transfectedor transformed with the vehicles and capable of expressing thepolypeptide. Such hosts may be prokaryotic, preferably bacteria, oreukaryotic, preferably yeast or mammalian cells. A preferred vectorsystem includes baculovirus expressed in insect cells. The DNA can beincorporated into host organisms by transformation, transduction,transfection, infection or related processes known in the art. Inaddition to DNA and mRNA sequences encoding the GP88 polypeptide, theinvention also provides methods for expression of the nucleic acidsequence. Further, the genetic sequences and oligonucleotides allowidentification and cloning of additional polypeptides having sequencehomology to the polypeptide GP88 described here.

[0123] An expression vector is a vector which (due to the presence ofappropriate transcriptional and/or translational control sequences) iscapable of expressing a DNA (or cDNA) molecule which has been clonedinto the vector and thereby produces a polypeptide or protein.Expression of the cloned sequence occurs when the expression vector isintroduced into an appropriate host cell. If a prokaryotic expressionvector is employed, then the appropriate host cell would be anyprokaryotic cell capable of expressing the cloned sequence. Similarly,if an eukaryotic expression system is employed, then the appropriatehost cell would be any eukaryotic cell capable of expressing the clonedsequence. Baculovirus vector, for example, can be used to clone GP88cDNA and subsequently express the cDNA in insect cells.

[0124] A DNA sequence encoding GP88 polypeptide or its functionalderivatives may be recombined with vector DNA in accordance withconventional techniques including blunt-ended or staggered ended terminifor ligation, restriction enzyme digestion to provide appropriatetermini, filling in cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and ligation with proper enzymeligases. Techniques for such manipulations are discussed in (35).

[0125] A nucleic acid molecule is capable of expressing a polypeptide ifit contains nucleotide sequences which contain transcriptional andtranslational regulatory information and such sequences are operablylinked to nucleotide sequences which encode the polypeptide. An operablelinkage is a linkage in which the regulatory DNA sequences and the DNAsequence sought to be expressed are connected in such a way as to permitgene expression. The precise nature of the regulatory regions needed forgene expression may vary from organism to organism but shall in generalinclude a promoter region, which in prokaryotes contains both thepromoter (which directs the initiation of RNA transcription) as well asthe DNA sequences which when transcribed into RNA will signal theinitiation of protein synthesis. Such regions will normally includethose 5′ non-coding sequences involved with the initiation oftranscription, translation such as the TATA box, capping sequence, CAATsequence and the like.

[0126] If desired, the 3′ non-coding region to the gene sequenceencoding the protein may be obtained by described methods (screeningappropriate cDNA library or PCR amplification). This region may beretained for the presence of transcriptional termination regulatorysequences such as termination and polyadenylation. Thus, by retainingthe 3′ region naturally contiguous to the DNA sequence coding for theprotein, the transcriptional termination signals may be provided. Wherethe transcription termination signals are not provided or satisfactorilyfunctional in the expression host cells, then a 3′ region from anothergene may be substituted.

[0127] Two DNA sequences such as a promoter region sequence and GP88encoding sequence are said to be operably linked if the nature of thelinkage between the sequences does not (1) result in the introduction ofa frame-shift mutation or (2) interfere with the ability of the promotersequence to direct transcription of the polypeptide gene sequence.

[0128] The promoter sequences may be prokaryotic, eukaryotic or viral.Suitable promoters are inducible, repressible or constitutive. Examplesof suitable prokaryotic promoters are reviewed by (44-46).

[0129] Eukaryotic promoters include but are not limited to the promoterfor the mouse methallothionein I gene (47), the TK promoter of HerpesVirus (48), the gene gal4 promoter (49), the SV40 early promoter (50),the mouse mammary tumor virus (MMTV) promoter, and the cytomegalovirus(CMV) promoter (51). Strong promoters are preferred. Examples of suchpromoters are those which recognize the T3, SP6 and T7 polymerases, thePL promoter of bacteriophage lambda, the recA promoter, the promoter ofthe mouse methallothionein I gene, the SV40 promoter and the CMVpromoter.

[0130] It is to be understood that application of the teachings of thepresent invention to a specific problem or environment will be withinthe capability of one having ordinary skill in the art in light of theteachings contained herein. The present invention is more fullyillustrated by the following non-limiting examples.

EXAMPLE 1 Isolation of PC Cell Line

[0131] The role of autocrine growth factor production in the loss ofdifferentiation ability and acquisition of tumorigenic properties inmammalian cells has been studied using a murine model system developedby the inventor. It consists of the mouse C3H adipogenic cell line 1246(9), a series of cell lines which are differentiation-deficient and haveincreasing tumorigenic properties. 1246 cells proliferate anddifferentiate in a serum-free defined medium (9). In defined medium,1246 cells stringently require insulin for proliferation and fordifferentiation (10). Insulin-like growth factor I (IGF-I) can replaceinsulin for proliferation but not for differentiation. From 1246 cellsmaintained in the absence of insulin, insulin-dependent cell lines wereisolated (11).

[0132] 1246-3A was particularly studied. 1246-3A cells had lost theability to differentiate and had become tumorigenic in vivo (11).1246-3A cells formed tumors when 10⁶ cells were injected into syngeneicC3H mice within 6 weeks, whereas 1246 cells were non-tumorigenic. By anin vitro-in vivo shuttle technique, highly tumorigenicinsulin-independent cell lines were subsequently isolated and analyzed(12).

[0133] The shuttle technique consisted of subcutaneously injecting1246-3A cells (10⁶ cells/mouse) into syngeneic C3H mice. The tumorresulting from the injection of the cells was then minced and plated inprimary culture into defined medium deprived of insulin (DME-F12nutrient medium 1:1 mixture supplemented with fibronectin, transferrinand FGF). Cells that had started to grow were subcultured when theyreached confluency to be: (1) either frozen in the presence of 10% FetalBovine serum and 10% Dimethylsulfoxyde (DMSO) for long termconservation; (2) injected subcutaneously into C3H mice at differentcell densities (10⁶, 10⁵, 10⁴ cells/mouse). Rate of appearance of tumorand size of tumor was monitored. Tumors that appeared were again putback in culture in the insulin-free medium. Cells growing in theseconditions were reinjected back into the animal.

[0134] By this in vitro-in vivo shuttle technique insulin-independentcells with increasing tumorigenic properties were obtained. Inparticular, the highly tumorigenic cell line named PC was isolated (12).PC cells can form tumors even when 10⁴ cells are injected subcutaneouslyinto syngeneic C3H mice.

[0135] The PC cell line has at least the following characteristics:

[0136] (1) These cells represent an excellent model system fortumorigenicity studies: these cells can proliferate in a simple definedmedium DME-F12 nutrient mixture supplemented with 2 μg/ml fibronectinand 10 μg/ml transferrin, and they can be injected into syngeneic hostsand do not require the use of nude mice which are expensive andnecessitate special handling.

[0137] (2) When the PC cells reach confluency, the cells can bemaintained in a complete serum-free and factor-free medium. Their growthis maintained solely by the nutrient medium and the factors that thecells secrete in their conditioned medium, thus conditioned medium is agood source for characterization and purification of factors requiredfor proliferation of tumor cells.

EXAMPLE 2 GP38 is an Autocrine Growth Factor for the Highly TumorigenicPC Cells.

[0138] It was shown that PC cell conditioned medium contained growthpromoting activity that was purified by chromatographic techniques (4).The purified factor called GP88 precursor was sequenced and shown to besimilar to the epithelin/granulin precursor.

[0139] Experiments were then carried out to examine whether theproduction of GP88 by PC cells stimulated their growth in an autocrinefashion. For this purpose, PC cells were cultivated in the presence ofGP88 antibody that can neutralize GP88 activity. DNA synthesis of PCcells was measured in the presence of increasing amounts of either thenon-immune IgG or the anti-K19T IgG.

[0140] As shown in FIG. 2, the addition of anti-GP88 IgG inhibited PCcell growth in a dose-dependent manner, directly demonstrating that GP88production by the PC cells is required for their growth. Non-immune IgGhad no effect. Here, PC cells were plated in 96-well plates at a densityof 2×10⁴ cells/well in DME/F12 medium supplemented with 2 μg/mlfibronectin and 10 μg/ml transferrin (2F medium). After 6 hours when thecells were attached, anti-GP88 IgG fraction was added. 36 hours later,³H-thymidine (0.25 μCi/ml) was added for an additional 8 hours. Cellswere collected by trypsinization on glass filters by a cell harvesterand the radioactivity corresponding to ³H-thymidine incorporated intoDNA counted by liquid scintillation counter. Values (FIG. 2) areexpressed as % of control corresponding to thymidine incorporation in PCcells treated with equivalent amounts of nonimmune IgG.

[0141] Similar results were obtained using anti K19T and anti E19Vmonoclonal antibodies. Anti K19T monoclonal antibody inhibited thegrowth of PC cells and of rat leukemia cells in a dose dependentfashion. Moreover, anti E19V monoclonal antibody inhibited the growth ofthe human breast carcinoma cell line MCF7, with an ED₅₀ of 100 μg/ml.

EXAMPLE 3 Expression of GP88 in the 1246, 1246-3A and PC Cell Lines

[0142] Since GP88 protein was purified from PC cell conditioned medium,experiments were carried out to compare the expression of GP88 mRNA andprotein in the three cell lines.

[0143] Comparative tumorigenicity studies of 1246, 1246-3A and PC celllines in C3H mice showed that PC cells are highly tumorigenic whencompared to 1246-3A cells since they can develop tumors when 10⁴cells/mouse are injected into C3H mice. 1246-3A cells make tumors wheninjected a 10⁶ cells/mouse, whereas in syngeneic hosts, 1236 cells arenon-tumorigenic (12).

[0144] The following methods were used for the studies of comparing thelevel of GP88 in the model system consisting of the three cell lines1246, 1246-3A and PC.

[0145] Cell Culture

[0146] 1246 stock cells were maintained in DME/F12 nutrient medium (1:1mixture of Dulbecco's modified Eagle medium and Ham's nutrient F12)supplemented with 10% fetal bovine serum (FBS). 1246-3A and PC stockcells were maintained in defined media. For PC stock cells, it consistedof DME-F12 medium supplemented with 2 μg/ml of human plasma fibronectinand 10 μg/ml of human plasma transferrin (2F medium). For 1246-3A cells,the defined medium called 3F medium consisted of DME-F12 mediumsupplemented with 2 μg/ml of human plasma fibronectin and 10 μg/ml ofhuman plasma transferrin and 1 ng/ml of basic fibroblast growth factor(bFGF). For comparative studies the three cell lines were plated inDME-F12 medium supplemented with 2% FBS.

[0147] RNA Isolation and Northern Blot Analysis

[0148] For Northern blot analysis of GP88 mRNA expression in rodentcells or tissues (mouse and rats), we used a mouse GP88 cDNA probe 311bp in length starting at nucleotide 551 to 862 (corresponding toamino-acid sequence 160 to 270). The probe was ³²P-labeled byrandom-priming reaction.

[0149] Total cellular RNA was isolated by RNAzol solution (Cinnabiotech)or Trizol solution (Life Technologies) based on a modification of thesingle step guanidinium isothiocyanante/phenol chloroform method (52).

[0150] Fifteen or twenty micrograms of total RNA per sample weresubjected to electrophoresis on a denaturing 1.2% agarose gel containing0.22 M formaldehyde in 1× MOPS (10× MOPS: 0.2 M MOPS, 50 mM NaOAc 10 mMEDTA). RNA was blotted on nitrocellulose membrane (MSI Inc., Westboro,Mass.) by overnight capillary transfer in 10× SSC (20× SSC =3M NaCl,0.3M Na Citrate pH 7.0). The filters were baked at 80° C. under vacuumfor 2 hrs and then prehybridized at 42° C. for 4 hrs in hybridizationsolution consisting of 50% formamide, 5×SSPE (1×SSPE=0.16 M sodiumchloride, 50 mM sodium phosphate pH 7.4, 1 mM EDTA), 1% SDS,5×Denhardt's solution (1×Denhardt's solution=0.02% each ofpolyvinylpirrolidone, Ficoll and bovine serum albumin), 1 μg/ml poly-Aand 100 μg/ml denatured salmon sperm DNA at 42° C. Hybridization wasperformed overnight at 42° C. in the same solution with 10⁶cpm/ml ofrandom-primed ³²P-labeled GP88 cDNA probe. Filters were washed twice for25 min at 42° C. in 2× SSC and 1% SDS, followed by two 15 min. washes at56° C. in 0.2× SSC and 1% SDS. Dried filters were exposed to Iodak XAR-5film (Iodak, Rochester, N.Y.) at −70° C. with an intensifying screen(Dupont, Boston, Mass.). Results were quantitated by densitometricscanning. Ribosomal protein L₃₂ mRNA was detected as internal standardfor normalizing RNA loading.

[0151] Preparation of Cell Lysate, Immunoprecipitation and Western BlotAnalysis

[0152] Cells in 10 cm culture dishes were washed once with PBS bufferand lysed on ice for 10 min. with 1 ml PBS buffer containing 1% TritonX-100. Cell lysate was sonicated for 10 seconds on ice, centrifuged at10,000×g for 10 min., then the supernatant was collected and stored at−70° C. until use.

[0153] Amounts of cell lysate and culture medium to be analyzed werenormalized by cell number of 18×10⁵ and 3×10⁵ respectively. The proteaseinhibitors: 200 μM PMSF, 1 μM leupeptin, 0.5 μM aprotinin, and 1 μM EDTAwere added per sample. Each sample was incubated at 4° C. for 4 hourswith 5 μg of affinity purified anti-K19T IgG conjugated to agarose withshaking. Precipitates were collected by centrifugation, washed threetimes with PBS buffer, resuspended in 20 μl SDS sample buffer containing5% β-mercaptoethanol, boiled for 5 min., and then separated by SDS-PAGEon 10% polyacrylamide gels according to the method of Laemmli (56).Proteins were electro transferred to immobilon membranes and GP88detected using anti K19T antibody conjugated to horseradish peroxidaseand detected by enhanced chemiluminescence (ECL).

[0154] When expression of GP88 mRNA was investigated in the three celllines cultivated in DME/F12 medium supplemented with 2% fetal bovineserum (FBS) our laboratory as a probe the results show that the highestlevel of mRNA expression for GP88 is found in the highly tumorigenic PCcells (FIG. 1B).

[0155] The levels of GP88 protein expression were examined by Westernblot analyses using anti-K19 antibody both in cell lysates and inculture medium of 1246, 1246-3A and PC cells as described above. Asshown in FIG. 1A, the level of GP88 was undetectable in the culturemedium of 1246 cells, 3T3, and 1246-3A cells and increased dramaticallyin the culture medium of the highly tumorigenic PC cells. The sameresults were obtained for GP88 expression in cell lysates.

[0156] GP88 expression was examined in 1246 cells in different cultureconditions such as defined medium and serum containing medium. It wasshown that the level of expression of GP88 mRNA (measured by Northernblot analysis) and protein (measured by Western blot analysis) in 1246and in 1246-3A cells is inhibited when the cells are treated with 2%fetal bovine serum indicating the presence of circulating inhibitors ofGP88 expression in fetal bovine serum (FIG. 1C). This inhibition of GP88expression was also observed when the activity of GP88 promoter linkedto a luciferase reporter gene was measured indicating that theseinhibitors are effective in inhibiting the transcriptional activity ofthe GP88 gene. Such inhibitors can be useful to develop GP88antagonizing agents which will be useful as anti-tumor or antiviraltherapy. We have also showed that GP88 mRNA expression is stimulated byEGF in the 1246 cells and by insulin in the human mammary carcinomacells MDA MB-453.

EXAMPLE 4 GP88 Expression and Biological Activity in Mammary EpithelialCells.

[0157] (a) Expression of GP88 in Breast Carcinoma Cells.

[0158] Experiments were carried out to examine the level of GP88expression in breast carcinoma and to examine if GP88 is a growth factorfor mammary epithelial cells. The rationale in support of thispossibility is: GP88 is a potent growth stimulator for mammaryepithelial cells (see next paragraph); receptors for GP88 (our studies)and processed form epithelin 1 (54) have been characterized on mammaryepithelial cells; breast carcinoma cell lines with different degrees ofhormonal dependency are available for study; and lastly there are agrowing number of reports emphasizing the importance of the insulin/IGFpathway in the growth control of mammary cells indicating that an escapefrom this regulation is occurring in malignant breast carcinomas (14,15). Since PC cells which display an over expression of GP88 areinsulin/IGF independent, this would support the rationale of GP88deregulation in human breast carcinoma.

[0159] We have investigated the level of expression of GP88 mRNA inthree well studied human breast carcinoma cell lines, MCF-7, which isestrogen receptor positive (ER+) , and two cell lines, MDA-MB-453 and468, being estrogen receptor negative (ER−). MDA-MB-468 has also beencharacterized as having a defective insulin and insulin-like growthfactor signaling (53). FIG. 5 shows that GP88 mRNA is expressed in thethree cell lines but that the level of expression is higher in theER-cell lines, MDA-MB-453 and 468, than in the ER+MCF7 cells, indicatingthat in human breast carcinoma, increased malignancy may be accompaniedby increased GP88 expression.

[0160] (b) Biological Activity of GP88 in Mammary Epithelial Cells

[0161] We investigated the effect of GP88 on the proliferation of avariety of cell lines including fibroblasts and mammary epithelialcells. We have found that GP88 had a profound growth promoting effect onthe mouse mammary epithelial cell line C57MG. As shown in FIG. 6, a5-fold increase in DNA synthesis was observed at a concentration of 150ng/ml (2 nM) either with GP88 purified from PC cells or with recombinantGP88 expressed in insect cells.

[0162] The ability of GP88 purified from PC cells (upper panel) andrecombinant GP88, rGP88 (lower panel), to stimulate DNA synthesis inC57MG cells was measured by incorporation of ³H-thymidine inserum-starved quiescent cells. Interestingly, in contrast to the growthpromoting effect of GP88 on breast epithelial cells, the 6 kDa epithelin2 (epi 2) has been reported as a growth inhibitor, at least forMDA-MB-468 cells when given at concentrations up to 100 nM (54). Thesedata suggest that the precursor, i.e., GP88, and the processed form,i.e., epi 2, have opposite effects on mammary epithelial cell growth.

EXAMPLE 5 Cloning of GP88 cDNA

[0163] GP88 protein purified from PC cell conditioned medium wassequenced after digestion with cyanogen bromide and trypsin. Sequencesof N-terminal regions and 6 peptides were obtained (4). Sense andantisense redundant oligonucleotide primers complementary to theobtained amino acid sequences were synthesized and used in thepolymerase chain reaction using the touch down PCR method with firststrand cDNA of PC cells as template. From the touch down PCR using aprimer pair SCV157 and ANG300, a 444 bp amplified product was obtained.This cDNA was then used to screen a lambda-ZAP cDNA library preparedfrom PC cells in our laboratory. One million unamplified plaques werescreened by plaque hybridization with ³²P-labeled PCR generated cDNAfragment. Positive clones were isolated by an additional 3 rounds ofplaque purification. Full length GP88 cDNA clone was obtained andsequenced. Full length cDNA was 2137 nucleotides in length with thefirst ATG located at 23 bp from the 5′ end, an open reading frame (ORF)1770 nucleotides long, and a 3′ untranslated region having a polyA tailat position 2127. The sequence was identical to the published mousegranulin (5) except for one nucleotide (T instead of G) at position 1071of GP88 cDNA (position 1056 of mouse granulin), which resulted in thechange of amino acid from arginine to leucine, and a nucleotidesubstitution at position 1483 with no change in amino acid (FIG. 8).This study demonstrated that GP88 is similar to epithelin/granulinprecursor and provided a cDNA to pursue our study of GP88 expression.

EXAMPLE 6 Expression of Mouse GP88 cDNA in Baculovirus

[0164] For recombinant GP88 production, the method of choice was toexpress GP88 in the baculovirus system. A full length GP88 cDNA(obtained by screening PC cell cDNA library) including the signalpeptide was ligated into the baculovirus transfer vector pVL1392 (inVitrogen, San Diego, Calif.). Plasmid pVL1392-GP88 was used toco-transfect Sf9 insect cells with baculovirus DNA. Recombinant virusesencoding GP88 were isolated and plaque purified. For infection andproduction of recombinant GP88, Sf9 cells were seeded in Grace's mediumcontaining 10% fetal bovine serum (FBS) in T75 cm² flasks. Afterinfection with recombinant baculovirus-GP88, insect cells weremaintained in Grace's medium for 48 hours at 27° C. Conditioned mediumwas collected by centrifugation and recombinant GP88 (rGP88) waspurified by a 2 step purification protocol consisting ofheparin-sepharose and immunoaffinity chromatography as described inExample 6. SDS-PAGE analysis of rGP88 indicated that rGP88 migratesfaster than PC cell derived GP88 corresponding to an apparent MW of 76kDa. N-terminal sequencing analysis of rGP88 indicated that it wasidentical to GP88 purified from PC-CM. The difference of molecularweight between GP88 and rGP88 is due to a difference in glycosylationstatus of GP88 in insect cells. As shown in FIG. 6, biological activityof rGP88 was identical to that of GP88 purified from PC cells,indicating that the different glycosylation status of GP88 in insectcells and mammalian cells did not affect the biological potency of theprotein.

[0165] The rGP88 produced from insect cells can be used for biological,and binding studies and to develop monoclonal antibodies to the intactGP88

EXAMPLE 7 Purification of GP88 and Recombinant GP88 by AffinityChromatography

[0166] The conditioned medium (2000 ml) from PC cells was diluted withthe same volume of H₂O and loaded on a 2.5 ml heparin-sepharose CL-6Bcolumn equilibrated in 10 mM sodium phosphate buffer pH 7.4 containing75 mM NaCl (Pharmacia, Uppsala, Sweden). The column was washed with atleast 10 bed volumes of the same equilibration buffer followed by a washwith 10 mM sodium phosphate buffer containing 0.15 M NaCl. The fractioncontaining GP88 was eluted with 5 bed volumes of 0.4 M NaCl, 10 mMTris-HCl, pH 7.5. The eluate was stored at −20 C. for furtherpurification. A synthetic peptide K19T (sequence: KVIAPRRLPDPQILIKSDT)was used to raise the antisera against the GP88 used in theimmunoaffinity step. The K19T peptide was linked to CNBr-activatedSepharose 4B according to the method provided by the manufacturer(Pharmacia, Uppsala, Sweden). The specific anti-K19 antibody waspurified using the K19T peptide affinity column by elution at acidic pH.Specifically, anti-K19T IgG was applied to a K19T peptide-Sepharose 4Bcolumn equilibrated with 10 mM sodium phosphate buffer pH 6.5 (Buffer A)at a flow rate of 0.8 ml/hr, and circulated at 4° C. overnight. Afterwashing the column with 7 ml of Buffer A, the conjugate was eluted with1 ml of HCl, pH 2.9, then 1 ml of HCl, pH 2.5 at a flow rate of about0.1 ml/min in a tube containing 0 1 ml of 1M sodium phosphate buffer pH7.0 to neutralize the pH. The concentration of affinity-purified IgG wasdetermined by the absorbance at 280 nm.

[0167] The purified Ab-K19T (1 mg) was then conjugated to 1 ml ofagarose beads (Sulfolink coupling gel, Pierce, Rockford, Ill.) usingprotocols provided by the manufacturer. The final coupled columncontained 600 μg anti-K19T/ml gel. The Ab-K19T agarose was packed in acolumn and washed extensively with PBS. The eluate from heparinsepharose CL-6B column was diluted with 3 volumes H₂O and loaded on theAb-K19T column. After washing the column with buffer consisting of 750mM NaCl in 10 mM NaPO4 pH 7.5, the fraction containing GP88 was elutedby elution buffer (150 mM NaCl, pH 2.5 (HCl)). To neutralize, 1/10volume (v/v) 1 M sodium phosphate pH 6.5 buffer was added to the eluateand the protein concentration was determined by amino acid analysis ormicro BCA kit (Bio-Rad, Richmond, Calif.). In general 50 μg of GP88 waspurified on a 350 μl column.

[0168] This method is also adequate for the purification of recombinantGP88 such as constructed in a baculovirus expression vector andexpressed in insect cells. This method is also adequate to purify humanGP88 using for the immunoaffinity step human GP88 antibody conjugated toadequate support (sepharose or agarose).

EXAMPLE 8 Development of Neutralizing Antibody for GP88

[0169] Peptides corresponding to various regions of mouse and human GP88were synthesized and conjugated to keyhole limpet Hemocyanin (KLH) bythe “glutaraldehyde method.” Peptide KLH conjugate was injected intochinchilla rabbits to raise anti-GP88 antibody. Two peptides, K19T andS14R, listed below, were found to generate neutralizing antibodies.Equivalent regions such as E19V of the human GP88 amino acid sequenceswere used to develop neutralizing anti-human GP88 monoclonal antibodies.Peptides were as follows: P12T from P208 to T219 PDAKTQCPDDST K19T fromK344 to T362 KKVIAPRRLPDPQILKSDT S14R from S562 to R575 SARGTKCLRKKIPRE19V (human GP88) EKAPAHLSLPDPQALKRDV A14R (human GP88) ARRGTKCLRREAPR

[0170] Properties of anti-sera are the following:

[0171] (1) Anti-K19T, anti-S14R and anti-E19V antibody recognize an 88kDa GP88 in conditioned medium of cells in culture, in cell lysates andin tissue extracts.

[0172] Tissue distribution of GP88 protein expression indicates that itis widely expressed and that most tissues express the unprocessedprecursor, i.e., GP88, rather than processed 6 kDa forms, i.e., epi 1and 2. GP88 is found in serum (mouse serum contains about 150 ng 88 kDaGP88/ml), and expressed in adipose tissue, in brain (molecular weightvaries between 45 and 88 kDa), in testes, in ovary, in liver and inkidney.

[0173] (2) Anti-K19T antibody is a neutralizing antibody. Anti-K19Tantibody neutralizes the biological effect of GP88 secreted by PC cellswhich is required for their proliferation. (FIG. 2). Addition ofanti-K19T IgG into culture medium of PC cells results in a dosedependent inhibition of PC cell growth. Non-immune IgG had no effect.Inhibition of cell proliferation of cells expressing GP88 has also beenobtained using monoclonal antibody K19T for PC cells, rat leukemia celllines and using monoclonal antibody E19V for neutralizing human GP88 inhuman breast carcinomas. This demonstrates that the E19V region of humanGP88 (and K19T region of mouse GP88) is a region of great biologicalimportance and that any antibody raised against this region will resultin obtaining a neutralizing antibody that can be used for therapy ofdiseases due to increased GP88 expression or increased responsiveness toGP88. The same is true for the S14R region of mouse GP88 and A14R regionof human GP88.

[0174] (3) Anti E19V monoclonal antibody is a neutralizing antibody. Wehave shown that at a dose of 100 μg/ml, E19V antibody inhibits thegrowth of the human breast carcinoma cell line MCF7 by 50% using³H-thymidine incorporation assay as described above for PC cells.

EXAMPLE 9 Growth and Tumorigenic Properties of Cells Transfected withExpression Vector Containing GP88 cDNA in Antisense Orientation.

[0175] The examples above demonstrate that GP88 is overexpressed by thehighly tumorigenic PC cell line. Since the cultivation of PC cells inthe presence of neutralizing anti-GP88 antibody had resulted in growthinhibition, it indicated that GP88 is required for the growth of PCcells. In order to test whether GP88 overexpression is responsible forthe high tumorigenic properties of PC cells in vivo, we examined thegrowth properties and the tumorigenic ability of PC cells transfectedwith a cytomegalovirus promoter controlled expression vector containingGP88 cDNA in antisense orientation in order to obtain high levels ofantisense RNA transcription. As control, we used PC cells transfectedwith empty vector.

[0176] A 228 bp fragment of GP88 cDNA was cloned in the antisenseorientation in pCMV4 expression vector (Andersson, S., et al, 1989) (51)containing CMV promoter and hGH transcription termination andpolyadenylation signals (pCMV4-GP88AS). PC cells were co-transfectedwith the 20 μg of antisense pCMV4-GP88AS and 2 μg of pRSVneo expressionvector containing the neomycin resistant gene by the calcium phosphatemethod. Control cells were co-transfected with empty pCMV4 vector andpRSVneo as described above. Transfected cells were selected in thepresence of neomycin. Neomycin resistant colonies were cloned and cellswere assayed first by detecting the presence of pCMV4-GP88AS by PCR.Twenty-four positive neomycin resistant clones containing the antisensepCMV4-GP88AS were isolated. Nine have been isolated and screened forexpression of the antisense transcript. Three clones were furthercharacterized. Western blot analysis of cell lysates and conditionedmedium using anti-GP88 antiserum (i.e., anti-K19T antibody) wasperformed in order to determine the level of GP88 expression intransfected antisense cells and control cells (FIG. 7). Culture mediumand cell lysates were prepared by immunoprecipitation with anti-K19Tantibody. Protein samples corresponding to 3×10⁶ cells/lane wereanalyzed by Western blotting with anti-GP88 antibody. The resultsindicate that GP88 levels are significantly lower in antisense, than incontrol, transfected cells particularly for AS1 and AS18 clones.

[0177] Stable Transfection of Antisense GP88 cDNA into PC Cells

[0178] PC cells were transfected with a 228 bp antisense cDNA fragmentof GP88 including start codon region obtained by digesting with Sma Iand Xba I GP88 cDNA clone and cloning the obtained cDNA fragment in theantisense orientation into Xba I and Sma I site of the mammalianexpression vector pCMV4 as shown in FIG. 11. The stable transfection ofPC cells was performed by the Calcium Phosphate method (55) in DMEmedium (Dulbecco's Modified eagle Medium) containing 3.7 g/L sodiumbicarbonate supplemented with 10% FBS. 2-4 hours prior to transfection,a calcium phosphate precipitate was made with 20 μg of plasmid pCMV4constructed with antisense GP88 cDNA, 2 μg of plasmid carrying neomycinresistance selectable marker (pRSVNEO), and 20 μg of pSK as carrier DNA.After 25 minutes, the precipitate was added dropwise to the cells. After7 hours, the medium was aspirated and the cells were shocked with 10%DMSO in PBS for 2-3 min., washed twice and fed with complete medium (DMEsupplemented with 10% FBS). One day after transfection, the cells weresplit 1:3 and selected for resistance to Geneticin (G-418 Sulphate,Gibco-BRL) at 400 μg/ml. Media was changed 2 days later to remove deadcells and every 3-4 days thereafter. 10-14 days after the 1:3 split,colonies were picked with a cloning ring and transferred into 48 wellplate, then passaged into 24 well plate, 12 well plate, and 60 mm dishsubsequently. The transfectants were analyzed or frozen. Co-transfectionof the empty pCMV4 vector and pRSVNEO was used for raising the emptyvector control transfectants.

[0179] After being selected by their ability to grow in the presence ofGeneticin , the transfected clones were analyzed by two assays asdescribed below:

[0180] The presence of the antisense cDNA construct is tested by PCRanalysis of genomic DNA of transfected clones using as primers anoligonucleotide located in the CMV promoter(5′-CCTACTTGGCAGTACATCTACGTA-3′) and the other corresponding to thestart codon of GP88 cDNA (S′ -CGAGAATTCAGGCAGACCATGTGGGTC-3′). Theseprimers would amplify a 551 bp DNA fragment from genomic DNA oftransfected cells containing the antisense DNA construct describedabove.

[0181] Then, the level of GP88 protein in antisense transfected cloneswas measured by Western blotting analysis of cell lysates andconditioned media collected from the transfected clones using anti-K19Tantibody to measure the efficacy by which the antisense GP88 inhibitedthe endogenous GP88 protein expression. Antisense clones that showed thehighest degree of inhibition of GP88 expression were selected foranalysis of their growth properties in vivo as described below. Analysisof GP88 expression in empty vector control transfected clones was donesimilarly.

[0182] The methods used in these various assays will now be described indetail.

[0183] PCR Selection of Transfectants

[0184] The presence of the antisense construct in the transfected cellswas determined by PCR analysis of their genomic DNA using as senseprimer SP647 (5′-CCTACTTGGCAGTACATCTACGTA-3′) corresponding to CMVpromoter region and antisense primer SP7(5′-CGAGAATTCAGGCAGACCATGTGGGTC-3′) corresponding to start codon regionof GP88. The sense primer SP647 and antisense primer AP912(5′-CTGACGGTTCACTAAACGAGCTC-3′) both located in the CMV4 promoter wereused to test whether CMV promoter was inserted into the genomic DNA ofcontrol transfectants which had been transfected with empty pCMV4vector.

[0185] For extracting genomic DNA for the PCR analysis, thetransfectants were lysed by buffer A (100 mM KCl, 10 mM Tris-HCl [pH8.3], 0.45% Tween 20 and 0.45% NP40) and proteinase K 120 μg/ml,incubated in 60° C. 1 hr, followed by boiling for 15 min. 50-100 ng DNAof each clone was used as template for PCR reaction. Non-transfected PCwere used as negative control. Constructed plasmid DNA was used aspositive control.

[0186] The PCR reaction was performed in 20 μl reaction mixturecontaining 10 mM Tris-HCl pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.1% TritonX-100, 0.2 mM dNTPs, 0.5 units Taq DNA polymerase, 3.2 pMol of eachprimer, and 50-100 ng of template DNA. The reaction tubes were heated to95° C. for 3 minutes, and then subjected to 40 cycles of 94° C. for 1minute, 55° C. for 2 minutes, and 72° C. for 3 minutes with a 10 minutes72° C. extension in a programmable thermal controller. Products wereanalyzed on a 1% agarose gel stained with ethidium bromide. The expectedsize of the amplified fragment corresponding to the presence of theantisense construct with the primers chosen was 551 bp.

[0187] Measurement of GP88 Protein Expression in Antisense and ControlTransfected clones

[0188] The function of transfecting antisense GP88 DNA in the cells isto inhibit endogenous GP88 expression. Therefore, the transfected clonesof cells which had been selected by the assays described above (i.e.,neomicin resistance and presence of antisense DNA in genomic DNA) wereexamined to determine the degree of inhibition of endogenous GP88expression. This was examined by measuring the level of GP88 in celllysates and in conditioned medium from antisense transfected clones andempty vector transfected control clones by immunoprecipitation andWestern blot analysis using anti-K19T antibody by the methods previouslydescribed. The transfected clones that displayed the highest degree ofinhibition of GP88 expression were further analyzed to examine theirgrowth properties in vitro and in vivo.

[0189] Cell Growth Assay

[0190] Cells were plated at a density of 3×10⁴ cells/well (12 wellplates, Corning) in 2 ml of DME/F-12 medium (1:1 mixture) supplementedwith 2% FBS or 2 μg/ml human fibronectin and 10 μg/ml human transferrin.Five days later, cells were washed with PBS, and cell number per wellwas determined by counting cells from duplicate wells using CoulterCounter after trypsinization.

[0191] The results showed that the antisense GP88 transfected PC clonesshowed a slower growth when cultivated either in serum containing mediumand in defined medium (2F medium) than the empty vector control PC cellscultured under equivalent conditions. These results are in agreementwith the fact that GP88 is required by the PC cells to proliferate.Since its expression is inhibited by the antisense, the growth of theantisense transfected cells is inhibited as a result.

[0192] Tumorigenicity Assay

[0193] Six weeks old female C3H mice were used for tumorigenicityassays. Sense/antisense or control transfectants were injectedsubcutaneously into syngeneic C3H mice at the density of 10⁶ cells peranimal. The appearance and size of tumors were examined. The mice werekilled 45-50 days after injection. Tumors were excised, their weightdetermined and the tumors were quick frozen and kept at −70° C.

[0194] In vivo tumorigenicity studies were are carried out by injecting5×10⁶ antisense and control cells (transfected with empty vector andnontransfected PC cells) subcutaneously in syngeneic C3H female mice.Mice were observed every day for appearance of tumors by scoring thetime of tumor appearance and the size of tumor. After 50 days, mice werekilled and blood was collected by heart puncture. Tumors were excised,weighed and either fixed for pathological examination or quick frozen inliquid nitrogen. Other organs of the mice were also collected andexamined.

[0195] Tumorigenicity for antisense clones and control clones wasexamined and compared to the wild type PC cells. Table 1 above, showsthe results obtained with one of them. FIG. 3 shows the picture of tumorbearing mouse detected with 10⁶ control transfected cells and of mouseinjected with antisense clone. Empty vector control transfected clonesmaintained similar tumorigenic properties as the parent PC cells,whereas no tumor formation was observed in the mice injected withantisense clones. Even after 90 days, these mice still did not presenttumors. This experiment was repeated twice. Moreover, additional clones(2 antisense and 2 control) were also examined. The same results wereobtained with other antisense clones.

[0196] Antisense cDNA for Human GP88

[0197] The approach for stable transfection of antisense GP88 cDNA in PCcells described above has also been applied to inhibiting GP88expression in human breast carcinoma cell lines.

[0198] In this case, the human antisense cDNA construct consisted of a400 bp cDNA fragment inserted in the antisense orientation in thecommercially available pcDNA3 mammalian expression vector (In Vitrogen,San Diego, Calif.) which contains pCMV4 cytomagelovirus CMV promoter andneomycin resistant gene so that double transfection of pCMV4 and pRSVneois not required like the ones described above for PC cells.

[0199] To generate the antisense cDNA fragment the following pairs ofprimers with appropriate restriction enzymes sites were synthesized andused in the PCR reaction: A-hGP88: 5′-A10GGATCCACGGAGTTGTTACCTGATC-3′(position nt: 362-344) H-hGP88: 5′-A10GAATTCGCAGGCAGACCATGTGGAC-3′(position nt: −12 to +8)

[0200] The amplified cDNA fragment with EcoRI and BamHI restrictionsites was inserted in the antisense orientation in the EcoRI and BamHIsites of the pcDNA3 mammalian expression vector. This expression vectorconstruct called pCAS was transfected in the human mammary carcinomacell fine MCF7 and MDA-MB-468 DME-F12 medium supplemented with fetalbovine serum by the calcium phosphate method (55). Selection oftransfected cells was done by cultivating the cells in the presence of800 μg/ml of Geneticin to select cells that are neomicin resistant.Neomicin resistant clones were picked with cloning rings and passaged inmedium supplemented with 10% fetal bovine serum (FBS) and with geneticin(800 μg/ml). Transfected clones selected for their resistance togeneticin were further examined by methods similar to the ones describedabove for PC cells transfected with mouse GP88 antisense cDNA. Thepresence of the antisense cDNA construct in genomic DNA was checked byPCR analysis using as primers for the PCR reaction T7 primer in thepCDNA3 expression vector and H-hGP88 primer described above. PCRreaction amplified a 420 bp DNA fragment in cells that expressed thetransfected human GP88 antisense DNA fragment.

[0201] Expression of endogenous GP88 was determined by Western blotanalysis of cell lysates and conditioned medium of transfected clonesusing anti E19V antiserum to select antisense clones with maximuminhibition of GP88 expression. Selected antisense clones were furtheranalyzed to examine their growth properties in vitro and in vivo.Transfection of empty pCDNA3 vector in MCF7 and MDA-MB-468 cells wasperformed as control for these experiments.

[0202] An alternative method to transfection of antisense cDNA is to useantisense oligonucleotides. It is known in the art that sequences aroundthe translation initiation site (ATG encoding the first methionine)provide good sequences for efficient antisense activity. Secondly,sequences with an adequate GC content and that start with either a G ora C have increased efficiency and stability in forming a hybrid withcorresponding sense sequence (32, 37, 38). Based on this rationale, itis anticipated that the following two sequences will be efficacious asantisense oligonucleotides to human GP88. The first one is a 22-mernamed HGPAS 1 starting 11 nucleotides upstream of the first ATG(methionine codon): HGPAS1 (22): 5′-GGGTCCACATGGTCTGCCTGC-3′. The secondoligomer is a 24 mer named HGPAS2 (24) located 21 nucleotides 3′(downstream) of the first ATG: HGPAS2(24):5′-GCCACCAGCCCTGCTGTTAAGGCC-3′. Other oligonucleotide antisensesequences can be explored by those of ordinary skill given the teachingsherein. To judge the efficacy of a sequence to inhibit GP88 expression,oligonucleotides will be added to breast carcinoma cells in culture orany other human cell types under study in increasing doses. Cells willbe collected at various time points (12 hours to several days) tomeasure the level of expression of GP88 protein by Western blot analysisor EIA using an antihuman GP88 antibody, using techniques known to thoseof ordinary skill in the art. Control cells will be treated with anonsense or a sense oligomer.

EXAMPLE 10 Inhibition of Tumor Growth in Humans

[0203] The present example provides the following:

[0204] Comparison of the expression of GP88 mRNA and protein in nontumorigenic mammary epithelial cells MCF10A and in malignant MCF7 and inMDA-MB-468 cells.

[0205] Growth of malignant MCF7 and MDA-MB-468 cells in the presence ofneutralizing anti-human GP88 antibody (anti-human E19V monoclonalantibody) leads to inhibition of proliferation of cells.

[0206] Proliferation in vitro and tumorigenicity in vivo of human breastcarcinoma cells are inhibited by inhibiting GP88 expression by antisenseGP88 DNA.

[0207] Comparison of GP88 Expression in Non tumorigenic Human MammaryEpithelial Cells and in Tumorigenic Breast Carcinoma Cell Lines

[0208] We investigated the expression of GP88 mRNA and protein in threehuman breast cell lines: The MCF10A cell line, which is a nontumorigenic human mammary epithelial cell line, and in two human breastcarcinoma cell lines, MCF7 which is estrogen receptor positive andMDA-MB-468 which is estrogen receptor negative.

[0209] Expression of GP88 mRNA expression was done by Northern blotanalysis of total RNA extracted from these cell lines using aradiolabeled human GP88 cDNA probe. Expression of GP88 protein wasmeasured by Western blot analysis of immunoprecipitated GP88 usingeither rabbit anti-human GP88 polyclonal antibody or the anti-human E19Vmonoclonal antibody. This latter antibody was developed by immunizingmice with human peptide E19V conjugated to protein as described inExample 8.

[0210] Anti-human GP88 antibodies that we have now at our disposal are:polyclonal antihuman GP88 antibody developed in rabbits using as antigena 37 kDa fragment of human GP88 expressed as a histidine tagged proteinin bacteria, and the anti-human GP88 antibody (polyclonal andmonoclonal) developed by using as antigen E19V peptide conjugated tokeyhole limpet hemocyanin. Development of these antibodies (polyclonaland monoclonal) are described above. All of these antibodies can be usedfor immunoprecipitation and Western blot analysis of human GP88 in humantissues and cells.

[0211] The Northern blot analysis shows that the expression of GP88 mRNAin the non tumorigenic MCF10A cells is very low and increases at least5-10 times in the human breast carcinoma cell lines MCF7 and MDA-MB-468(FIG. 14).

[0212] The results of the Western blot analysis of the three cell linesshow that GP88 protein expression is undetectable in culture medium (CM)collected from the non-tumorigenic MCF10A cells, whereas it increased10-20 times in media conditioned by the MCF7 cells and by the MDA-MB-468cells. In addition to being secreted in the culture medium of the breastcarcinoma cells, GP88 protein is also expressed at high levels in celllysates of the maligant cells, whereas it is undetected in the nontumorigenic MCF10A cells.

[0213] These data confirm that human breast carcinoma cells overexpressGP88 when compared to non tumorigenic human mammary cells.

[0214] Inhibition of Growth of Malignant MCF7 and MDA-MB-468 Cells inthe Presence of Neutralizing Anti-human GP33 Antibody (Anti-human E19VMonoclonal Antibody)

[0215] Experiments were carried out in which human breast carcinoma MCF7cells were incubated with different doses of anti-human GP88 monoclonalantibody (anti-E19V peptide IgG fraction). Proliferation was measured by[³H] thymidine incorporation into DNA. Control cells consisted of cellsincubated with the same amount of unrelated mouse IgG. Results show thatincubation of MCF7 cells with 25 μg/ml of anti-GP88 monoclonal antibodyleads to a 50% inhibition of thymidine incorporation, whereas 100 pg/mlresulted in an 80% inhibition of proliferation. Similar results werefound as indicated above for MDA-MB-468 cells. These data again confirmthat (i) anti-human E19V antibody neutralizes human GP88, and (ii)malignant human cells which secrete high levels of GP88 in culturemedium and which require GP88 to proliferate can be effectivelyinhibited by treatment with anti-human GP88 neutralizing antibody, thusdemonstrating that inhibition of GP88 action on tumor cells is aneffective therapy for inhibiting tumor growth of cells overexpressingGP88.

[0216] Proliferation in Vitro and Tumorigenicity in Vivo of Human BreastCarcinoma Cells are Inhibited by Inhibiting GP88 Expression by AntisenseGP88 DNA

[0217] Human breast carcinoma MDA-MB-468 cells were stably transfectedwith antisense human GP88 cDNA construct (400 bp fragment inserted intopCDNA3 expression vector). This construct is described in Example 9.Stable antisense clones were isolated and characterized. In particular,antisense clones were selected based on the fact that expression of GP88was effectively inhibited. This was determined by measuring GP88expression in antisense cells and empty vector control cells by Westernblot analysis of cell extracts and conditioned medium using antiGP88antibody. Several clones were obtained and characterized. Similarresults were obtained with all antisense clones isolated. Data presentedhere concern one antisense clone called 468AS. As shown in FIG. 15,transfection of antisense GP88 cDNA in MDA-MB-468 cells (468AS) resultedin inhibition of GP88 protein expression when compared to empty vectorcontrol MDA-MB-468 cells (468 Cont).

[0218] Measurement of the proliferation rate of antisense GP88 and emptyvector control cells indicated that antisense cells had an 80%inhibition of cell proliferation when compared to the empty vectortransfected cells. Our data with all the clones analyzed also showedthat the extent of growth inhibition was directly correlated to thedegree of inhibition of expression of GP88 in the antisense clones.

[0219] Antisense human breast 468AS cells which displayed the highestinhibition of GP88 expression, and empty vector control (468 Cont) cellswere injected subcutaneously into female nude mice in the breast area ata density of 2×10³ cells/mouse using 4 mice per cell line. Tumorappearance was monitored by visually inspecting the mice. After 4 weeks,the mice were sacrificed, tumors were excised and weighed (see Table 3below). TABLE 3 Effect of inhibition of GP88 expression by antisenseGP88cDNA transfection on tumor growth of human breast cancer cellsMDA-MB-468 in nude mice Cells Mice bearing tumors Weight of tumors (g)468 Cont empty 4/4 0.2 ± 0.06 vector 468AS antisense GP88 3/4   0.05 ±0/025**

[0220] The results show that inhibition of GP88 expression resulted in a75% inhibition of tumor growth for human breast carcinoma cells.

[0221] This experiment demonstrated that inhibition of GP88 expressionin human breast carcinoma cell lines leads to inhibition oftumorigenicity.

EXAMPLE 11 Diagnostic Test for Tumorigenicity

[0222] In teratoma and in breast cancer, an increase in tumorigenicproperties is associated with an increase in GP88 expression or anincrease in GP88 responsiveness.

[0223] Moreover, FIG. 4 shows that the level of expression of GP88 intumor tissue is increased when compared to the surrounding tissues.Accordingly, increase of GP88 level can be used as a diagnostic approachto detecting tumor. In human tumor biopsies, a change (increase) in GP88expression when compared to the level of GP88 in normal correspondingtissues is indicative of the state of tumorigecicity or malignancy ofthe tissue biopsy analyzed. Increase in expression of GP88 can bemeasured at the mRNA level or at the protein level. GP88 mRNA expressioncan be measured either by Northern blot analysis, RNAse protection assayor RT-PCR.

[0224] GP88 protein expression is quantitated by ELISA, EIA or RIA usingan anti-GP88 antibody.

[0225] The ability to measure GP88 expression in tissue extracts incomparison to corresponding tissues from normal subject can be used topredict the degree of tumorigenicity of a particular cancer or todetermine whether this particular cancer will be responsive to anti-GP88therapy.

[0226] For diagnosis of diseases associated with increase in GP88responsiveness, tissue biopsies to be analyzed will be treated withanti-GP88 neutralizing antibodies or anti-GP88 receptor antibodies tosee if such treatment inhibits growth of the cells. Alternatively, theability of GP88 antisense oligonucleotides to inhibit growth indicatesthat expression of GP88 is required for growth in vivo.

EXAMPLE 12 Characteristics of GP88 Cell Surface Receptors

[0227] Binding of iodinated GP88 to a variety of cell lines CCL64, 1246,PC and the mammary epithelial C57MG was determined in order to examinethe biochemical characteristics of GP88 cell surface receptors using themethods described below. For these studies we used affinity purifiedrecombinant GP88 (rGP88) from the culture medium of baculovirus infectedSF9 insect cells as ligand. Methods to prepare rGP88 have previouslybeen described.

[0228] a) Iodination of GP88.

[0229] Recombinant GP88 (rGP88) was iodinated by the chloramine T methodat 4° C. Other known methods can also be applied. Briefly, 1 μg of GP88was incubated for 2 min with ¹²⁵I Na (100 μCi) that had beenpreincubated for 90 seconds with 2 μg chloramine T. The reaction wasquenched with the addition of 100 μl saturated tyrosine, 100 μl of a 1%solution of bovine serum albumin (BSA) and 2 μg of sodium metabisulfite.After addition of 100 μl PBS, the iodinated protein was separated fromfree Na¹²⁵I by gel filtration on a Sephadex-G50 column that had beenpreincubated with PBS containing 1% bovine serum albumin and thenextensively washed with PBS to reduce non-specific binding. The labeledproteins were eluted with PBS and fractions monitored for radioactivity.Amount of incorporated radioactivity was estimated by TCA precipitation.Specific activity of ¹²⁵I-GP88 was typically 30-50 μCi/μg. This methodand other methods for iodination of proteins are well known to peopleskilled in the art can also be used to iodinate PC derived GP88 ratherthan rGP88 or any derivatives thereof.

[0230] b) ¹²⁵I-GP88 binding.

[0231] The example provided here describes binding assay with the minklung epithelial cell line CCL64 but has also been applied to severalcell lines including the 1246, PC cell lines and the mammary epithelialcell line C57MG. The binding assays were performed on cell suspension.CCL64 cells were cultivated as monolayer in DME medium supplemented with10% fetal bovine serum (FBS) until they reached confluency. At thattime, cells were washed with PBS and detached by incubation with asolution of 0.25 mg/ml of trypsin and 1 mM EDTA. The cells wereharvested by centrifugation, washed with culture medium and counted witha hemocytometer. For binding assays, 10⁶ cells were resuspended in 500μl of binding buffer consisting of DME medium supplemented with 1%bovine serum albumin in 1.5 ml ependorf tubes and incubated for 2 hoursat 25° C. with 10⁵ cpm of ¹²⁵I-rGP88 and increasing concentrations ofcold rGP88 from 1 to 100 ng/ml. Binding was stopped by centrifuging thecells followed by 3 successive washings at 4° C. with cold bindingbuffer followed by centrifugation. Cell pellets were counted with agamma counter. Scatchard analysis of binding data was carried out bycomputer Ligand program. Values correspond to the average of threeseparate experiments with duplicate determinations per experimentalpoint. Scatchard analysis of binding of ¹²⁵I-GP88 to CCL64 cells wascurvilinear corresponding to the presence of two classes of cell surfacereceptors: a high affinity class with a Kd of 4.3±1.5×10⁻¹¹ M, 560±170sites/cell and a low affinity class of receptors with a Kd of3.9±1.9×10⁻⁹M, 17,000±5900 sites/cell.

[0232] c) Cross-linking studies of ¹²⁵I-GP88 to CCL64 and other celllines.

[0233] Cross-linking of ¹²⁵I-GP88 to cell surface receptors was carriedout using disuccinimidyl suberate (DSS). For cross-linking studies,5×10⁵ cells were suspended in 250 μl of binding buffer in eppendorftubes. ¹²⁵I-GP88 was added in 50 μl of binding buffer (DME medium with1% BSA) with or without 100 fold excess unlabeled GP88 ligand. Bindingwas performed as described in the previous paragraph. At the end of theincubation period, the cells were washed one time with 0.2% BSA-DME andone time with PBS before cross-linking was carried out. Dissucinimidyesuberate (DSS) was dissolved in DMSO at a 100 mM just prior to use. Thecells were resuspended in 200 μl PBS containing 1 mM disuccinimidylsuberate (DSS,) at room temperature for 15 min. After crosslinking, thecells were centrifuged, washed and extracted with 25 μl extractionbuffer (PBS containing 1% Triton X-100, 0.1% SDS, 0.5 mMphenylmethylsulfonyl fluoride (PMSF) at 4° C. Samples were centrifugedfor 5 min at 13,000×g and 25 μl of supernatant from each sample wasmixed with 4 μl of 20% SDS and 15 μl 3× Laemmli's sample buffer (56)containing b-mercaptoethanol and boiled for 5 min. Electrophoresis ofsamples was carried out on 7% SDS polyacrylamide slab gel according toLaemmli (56) using a minigel apparatus (Bio-Rad, Richmond, Calif.). Thedried gels were exposed to X-ray films for autoradiography at −70° C.

[0234] As shown in FIG. 12, autoradiographic analysis revealed thepresence of one major cross-linked band with a molecular weight of190-195 kDa. This would correspond to a molecular weight for the unboundreceptor of about 110 kDa for the major band. The intensity of the majorcross-linked band was decreased in the lanes where binding was carriedout in the presence of excess cold GP88. Cross-linked band could not bedetected if experiment and gel electrophoresis was performed in theabsence of DSS. Additional experiments using samples treated or not withb-mercaptoethanol prior to performing the electrophoresis indicated thatthe cell surface receptors for epithelin/granulin precursor aremonomeric.

[0235] Cross-linking of ¹²⁵I-GP88 to cell surface receptors were alsocarried out with 3T3, PC cells and the mammary epithelial cells C57MG bythe same method. The results of these experiments indicated similarcross-linking pattern for GP88 in all cell lines tested suggesting thepresence of cell surface binding sites with similar size in fibroblasticand epithelial cells (FIG. 13)

[0236] GP88 Mediated Signal Transduction in Mammary Euithelial Cell LineC57MG

[0237] Experiments to determine the characteristics of the signaltransduction pathway activated by GP88 after binding to its cell surfacereceptors were carried out with GP88 in the mammary epithelial cell lineC57MG. We have shown that anti-K19 T antibody can immunoprecipitate thecomplex formed by GP88 and its cell surface receptors on various celltypes and in the mammary epithelial cells C57MG in particular. Thisfeature has allowed us to further characterize biochemical properties ofGP88 receptor and the signal transduction it mediates leading to growthstimulation. We have shown that GP88 receptors belong to the tyrosinekinase family of receptors. Upon binding of GP88 to its cell surface,GP88 receptor is activated by phosphorylation on tyrosine residuesresulting in phosphorylation of several signaling molecules includingIRS-1, SHC, Grb2 and leading to activation of MAP kinase ERK-2.

[0238] Having now fully described this invention, it will be appreciatedby those skilled in the art the same can be performed within a widerange of equivalent parameters concentration and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

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1 17 1 2137 DNA Mouse epithelin/granulin CDS (23)..(1789) The sequenceis identical to that of the published mouse granulin except for onenucleotide (T instead of G) at position 1071 of GP88 cDNA (position 1056of mouse granulin). 1 cggaccccga cgcagacaga cc atg tgg gtc ctg atg agctgg ctg gcc ttc 52 Met Trp Val Leu Met Ser Trp Leu Ala Phe 1 5 10 gcggca ggg ctg gta gcc gga aca cag tgt cca gat ggg cag ttc tgc 100 Ala AlaGly Leu Val Ala Gly Thr Gln Cys Pro Asp Gly Gln Phe Cys 15 20 25 cct gttgcc tgc tgc ctt gac cag gga gga gcc aac tac agc tgc tgt 148 Pro Val AlaCys Cys Leu Asp Gln Gly Gly Ala Asn Tyr Ser Cys Cys 30 35 40 aac cct cttctg gac aca tgg cct aga ata acg agc cat cat cta gat 196 Asn Pro Leu LeuAsp Thr Trp Pro Arg Ile Thr Ser His His Leu Asp 45 50 55 ggc tcc tgc cagacc cat ggc cac tgt cct gct ggc tat tct tgt ctt 244 Gly Ser Cys Gln ThrHis Gly His Cys Pro Ala Gly Tyr Ser Cys Leu 60 65 70 ctc act gtg tct gggact tcc agc tgc tgc ccg ttc tct aag ggt gtg 292 Leu Thr Val Ser Gly ThrSer Ser Cys Cys Pro Phe Ser Lys Gly Val 75 80 85 90 tct tgt ggt gat ggctac cac tgc tgc ccc cag ggc ttc cac tgt agt 340 Ser Cys Gly Asp Gly TyrHis Cys Cys Pro Gln Gly Phe His Cys Ser 95 100 105 gca gat ggg aaa tcctgc ttc cag atg tca gat aac ccc ttg ggt gct 388 Ala Asp Gly Lys Ser CysPhe Gln Met Ser Asp Asn Pro Leu Gly Ala 110 115 120 gtc cag tgt cct gggagc cag ttt gaa tgt cct gac tct gcc acc tgc 436 Val Gln Cys Pro Gly SerGln Phe Glu Cys Pro Asp Ser Ala Thr Cys 125 130 135 tgc att atg gtt gatggt tcg tgg gga tgt tgt ccc atg ccc cag gcc 484 Cys Ile Met Val Asp GlySer Trp Gly Cys Cys Pro Met Pro Gln Ala 140 145 150 tct tgc tgt gaa gacaga gtg cat tgc tgt ccc cat ggg gcc tcc tgt 532 Ser Cys Cys Glu Asp ArgVal His Cys Cys Pro His Gly Ala Ser Cys 155 160 165 170 gac ctg gtt cacaca cga tgc gtt tca ccc acg ggc acc cac acc cta 580 Asp Leu Val His ThrArg Cys Val Ser Pro Thr Gly Thr His Thr Leu 175 180 185 cta aag aag ttccct gca caa aag acc aac agc gca gtg tct ttg cct 628 Leu Lys Lys Phe ProAla Gln Lys Thr Asn Ser Ala Val Ser Leu Pro 190 195 200 ttt tct gtc gtgtgc cct gat gct aag acc cag tgt ccc gat gat tct 676 Phe Ser Val Val CysPro Asp Ala Lys Thr Gln Cys Pro Asp Asp Ser 205 210 215 acc tgc tgt gagcta ccc act ggg aag tat ggc tgc tgt cca atg ccc 724 Thr Cys Cys Glu LeuPro Thr Gly Lys Tyr Gly Cys Cys Pro Met Pro 220 225 230 aat gcc atc tgctgt tcc gac cac ctg cac tgc tgc ccc cag gac act 772 Asn Ala Ile Cys CysSer Asp His Leu His Cys Cys Pro Gln Asp Thr 235 240 245 250 gta tgt gacctg atc cag agt aag tgc cta tcc aag aac tac acc acg 820 Val Cys Asp LeuIle Gln Ser Lys Cys Leu Ser Lys Asn Tyr Thr Thr 255 260 265 gat ctc ctgacc aag ctg cct gga tac cca gtg aag gag gtg aag tgc 868 Asp Leu Leu ThrLys Leu Pro Gly Tyr Pro Val Lys Glu Val Lys Cys 270 275 280 gac atg gaggtg agc tgc cct gaa gga tat acc tgc tgc cgc ctc aac 916 Asp Met Glu ValSer Cys Pro Glu Gly Tyr Thr Cys Cys Arg Leu Asn 285 290 295 act ggg gcctgg ggc tgc tgt cca ttt gcc aag gcc gtg tgt tgt gac 964 Thr Gly Ala TrpGly Cys Cys Pro Phe Ala Lys Ala Val Cys Cys Asp 300 305 310 gat cac attcat tgc tgc ccg gca ggg ttt cag tgt cac aca gag aaa 1012 Asp His Ile HisCys Cys Pro Ala Gly Phe Gln Cys His Thr Glu Lys 315 320 325 330 gga acctgc gaa atg ggt atc ctc caa gta ggg tgg atg aag aag gtc 1060 Gly Thr CysGlu Met Gly Ile Leu Gln Val Gly Trp Met Lys Lys Val 335 340 345 ata gccccc ctc cgc ctg cca gac cca cag atc ttg aag agt gat aca 1108 Ile Ala ProLeu Arg Leu Pro Asp Pro Gln Ile Leu Lys Ser Asp Thr 350 355 360 cct tgtgat gac ttc act agg tgt cct aca aac aat acc tgc tgc aaa 1156 Pro Cys AspAsp Phe Thr Arg Cys Pro Thr Asn Asn Thr Cys Cys Lys 365 370 375 ctc aattct ggg gac tgg ggc tgc tgt ccc atc cca gag gct gtc tgc 1204 Leu Asn SerGly Asp Trp Gly Cys Cys Pro Ile Pro Glu Ala Val Cys 380 385 390 tgc tcagac aac cag cat tgc tgc cct cag ggc ttc aca tgt ctg gct 1252 Cys Ser AspAsn Gln His Cys Cys Pro Gln Gly Phe Thr Cys Leu Ala 395 400 405 410 cagggg tac tgt cag aag gga gac aca atg gtg gct ggc ctg gag aag 1300 Gln GlyTyr Cys Gln Lys Gly Asp Thr Met Val Ala Gly Leu Glu Lys 415 420 425 atacct gcc cgc cag aca acc ccg ctc caa att gga gat atc ggt tgt 1348 Ile ProAla Arg Gln Thr Thr Pro Leu Gln Ile Gly Asp Ile Gly Cys 430 435 440 gaccag cat acc agc tgc cca gta ggg caa acc tgc tgc cca agc ctc 1396 Asp GlnHis Thr Ser Cys Pro Val Gly Gln Thr Cys Cys Pro Ser Leu 445 450 455 aaggga agt tgg gcc tgc tgc cag ctg ccc cat gct gtg tgc tgt gag 1444 Lys GlySer Trp Ala Cys Cys Gln Leu Pro His Ala Val Cys Cys Glu 460 465 470 gaccgg cag cac tgt tgc ccg gcc ggg tac acc tgc aac gtg aag gcg 1492 Asp ArgGln His Cys Cys Pro Ala Gly Tyr Thr Cys Asn Val Lys Ala 475 480 485 490agg acc tgt gag aag gat gtc gat ttt atc cag cct ccc gtg ctc ctg 1540 ArgThr Cys Glu Lys Asp Val Asp Phe Ile Gln Pro Pro Val Leu Leu 495 500 505acc ctc ggc cct aag gtt ggg aat gtg gag tgt gga gaa ggg cat ttc 1588 ThrLeu Gly Pro Lys Val Gly Asn Val Glu Cys Gly Glu Gly His Phe 510 515 520tgc cat gat aac cag acc tgt tgt aaa gac agt gca gga gtc tgg gcc 1636 CysHis Asp Asn Gln Thr Cys Cys Lys Asp Ser Ala Gly Val Trp Ala 525 530 535tgc tgt ccc tac cta aag ggt gtc tgc tgt aga gat gga cgt cac tgt 1684 CysCys Pro Tyr Leu Lys Gly Val Cys Cys Arg Asp Gly Arg His Cys 540 545 550tgc ccc ggt ggc ttc cac tgt tca gcc agg gga acc aag tgt ttg cga 1732 CysPro Gly Gly Phe His Cys Ser Ala Arg Gly Thr Lys Cys Leu Arg 555 560 565570 aag aag att cct cgc tgg gac atg ttt ttg agg gat ccg gtc cca aga 1780Lys Lys Ile Pro Arg Trp Asp Met Phe Leu Arg Asp Pro Val Pro Arg 575 580585 ccg cta ctg taaggaaggg ctacagactt aaggaactcc acagtcctgg 1829 Pro LeuLeu gaaccctgtt ccgagggtac ccactactca ggcctcccta gcgcctcctc ccctaacgtc1889 tccccggcct actcatcctg agtcacccta tcaccatggg aggtggagcc tcaaactaaa1949 accttctttt atggaaagaa ggctctggcc aaaagccccg tatcaaactg ccatttcttc2009 cggtttctgt ggaccttgtg gccaggtgct cttcccgagc cacaggtgtt ctgtgagctt2069 gcttgtgtgt gtgtgcgcgt gtgcgtgtgt tgctccaata aagtttgtac gctttctgaa2129 aaaaaaaa 2137 2 589 PRT Mouse epithelin/granulin 2 Met Trp Val LeuMet Ser Trp Leu Ala Phe Ala Ala Gly Leu Val Ala 1 5 10 15 Gly Thr GlnCys Pro Asp Gly Gln Phe Cys Pro Val Ala Cys Cys Leu 20 25 30 Asp Gln GlyGly Ala Asn Tyr Ser Cys Cys Asn Pro Leu Leu Asp Thr 35 40 45 Trp Pro ArgIle Thr Ser His His Leu Asp Gly Ser Cys Gln Thr His 50 55 60 Gly His CysPro Ala Gly Tyr Ser Cys Leu Leu Thr Val Ser Gly Thr 65 70 75 80 Ser SerCys Cys Pro Phe Ser Lys Gly Val Ser Cys Gly Asp Gly Tyr 85 90 95 His CysCys Pro Gln Gly Phe His Cys Ser Ala Asp Gly Lys Ser Cys 100 105 110 PheGln Met Ser Asp Asn Pro Leu Gly Ala Val Gln Cys Pro Gly Ser 115 120 125Gln Phe Glu Cys Pro Asp Ser Ala Thr Cys Cys Ile Met Val Asp Gly 130 135140 Ser Trp Gly Cys Cys Pro Met Pro Gln Ala Ser Cys Cys Glu Asp Arg 145150 155 160 Val His Cys Cys Pro His Gly Ala Ser Cys Asp Leu Val His ThrArg 165 170 175 Cys Val Ser Pro Thr Gly Thr His Thr Leu Leu Lys Lys PhePro Ala 180 185 190 Gln Lys Thr Asn Ser Ala Val Ser Leu Pro Phe Ser ValVal Cys Pro 195 200 205 Asp Ala Lys Thr Gln Cys Pro Asp Asp Ser Thr CysCys Glu Leu Pro 210 215 220 Thr Gly Lys Tyr Gly Cys Cys Pro Met Pro AsnAla Ile Cys Cys Ser 225 230 235 240 Asp His Leu His Cys Cys Pro Gln AspThr Val Cys Asp Leu Ile Gln 245 250 255 Ser Lys Cys Leu Ser Lys Asn TyrThr Thr Asp Leu Leu Thr Lys Leu 260 265 270 Pro Gly Tyr Pro Val Lys GluVal Lys Cys Asp Met Glu Val Ser Cys 275 280 285 Pro Glu Gly Tyr Thr CysCys Arg Leu Asn Thr Gly Ala Trp Gly Cys 290 295 300 Cys Pro Phe Ala LysAla Val Cys Cys Asp Asp His Ile His Cys Cys 305 310 315 320 Pro Ala GlyPhe Gln Cys His Thr Glu Lys Gly Thr Cys Glu Met Gly 325 330 335 Ile LeuGln Val Gly Trp Met Lys Lys Val Ile Ala Pro Leu Arg Leu 340 345 350 ProAsp Pro Gln Ile Leu Lys Ser Asp Thr Pro Cys Asp Asp Phe Thr 355 360 365Arg Cys Pro Thr Asn Asn Thr Cys Cys Lys Leu Asn Ser Gly Asp Trp 370 375380 Gly Cys Cys Pro Ile Pro Glu Ala Val Cys Cys Ser Asp Asn Gln His 385390 395 400 Cys Cys Pro Gln Gly Phe Thr Cys Leu Ala Gln Gly Tyr Cys GlnLys 405 410 415 Gly Asp Thr Met Val Ala Gly Leu Glu Lys Ile Pro Ala ArgGln Thr 420 425 430 Thr Pro Leu Gln Ile Gly Asp Ile Gly Cys Asp Gln HisThr Ser Cys 435 440 445 Pro Val Gly Gln Thr Cys Cys Pro Ser Leu Lys GlySer Trp Ala Cys 450 455 460 Cys Gln Leu Pro His Ala Val Cys Cys Glu AspArg Gln His Cys Cys 465 470 475 480 Pro Ala Gly Tyr Thr Cys Asn Val LysAla Arg Thr Cys Glu Lys Asp 485 490 495 Val Asp Phe Ile Gln Pro Pro ValLeu Leu Thr Leu Gly Pro Lys Val 500 505 510 Gly Asn Val Glu Cys Gly GluGly His Phe Cys His Asp Asn Gln Thr 515 520 525 Cys Cys Lys Asp Ser AlaGly Val Trp Ala Cys Cys Pro Tyr Leu Lys 530 535 540 Gly Val Cys Cys ArgAsp Gly Arg His Cys Cys Pro Gly Gly Phe His 545 550 555 560 Cys Ser AlaArg Gly Thr Lys Cys Leu Arg Lys Lys Ile Pro Arg Trp 565 570 575 Asp MetPhe Leu Arg Asp Pro Val Pro Arg Pro Leu Leu 580 585 3 19 PRT mousegranulin PEPTIDE (1)..(19) Internal peptide of mouse GP88 used to raisethe antisera against the GP88 used in the immunoaffinity step. 3 Lys LysVal Ile Ala Pro Arg Arg Leu Pro Asp Pro Gln Ile Leu Lys 1 5 10 15 SerAsp Thr 4 12 PRT mouse granulin PEPTIDE (1)..(12) Internal peptide ofmouse GP88 used to raise the antisera against the GP88 used in theimmunoaffinity step. 4 Pro Asp Ala Lys Thr Gln Cys Pro Asp Asp Ser Thr 15 10 5 14 PRT mouse granulin PEPTIDE (1)..(14) Internal peptide of mouseGP88 used to raise the antisera against the GP88 used in theimmunoaffinity step. 5 Ser Ala Arg Gly Thr Lys Cys Leu Arg Lys Lys IlePro Arg 1 5 10 6 19 PRT Human granulin PEPTIDE (1)..(19) Internalpeptide of human GP88 used to develop neutralizing anti-human GP88monoclonal antibody. 6 Glu Lys Ala Pro Ala His Leu Ser Leu Pro Asp ProGln Ala Leu Lys 1 5 10 15 Arg Asp Val 7 14 PRT Human granulin PEPTIDE(1)..(14) Internal peptide of human GP88 used to develop neutralizinganti-human GP88 monoclonal antibody. 7 Ala Arg Arg Gly Thr Lys Cys LeuArg Arg Glu Ala Pro Arg 1 5 10 8 24 DNA mammalian primer (1)..(24)Internal peptide of CMV promoter used as PCR primer. 8 cctacttggcagtacatcta cgta 24 9 27 DNA mammalian primer (1)..(27) GP88 cDNA startcodon used as oligonucleotide PCR primer. 9 cgagaattca ggcagaccatgtgggtc 27 10 27 DNA mammalian primer (1)..(27) Antisense primeroligonucleotide primer 10 cgagaattca ggcagaccat gtgggtc 27 11 23 DNAmammalian primer (1)..(23) Antisense primer oligonucleotide primer 11ctgacggttc actaaacgag ctc 23 12 25 DNA mammalian primer (1)..(25) primer12 ggatccacgg agttgttacc tgatc 25 13 25 DNA mammalian primer (1)..(25)oligonucleotide PCR primer 13 gaattcgcag gcagaccatg tggac 25 14 21 DNAmammalian primer (1)..(21) Antisense oligonucleotide to human GP88 14gggtccacat ggtctgcctg c 21 15 24 DNA mammalian primer (1)..(24)Antisense oligonucleotide to human GP88 15 gccaccagcc ctgctgttaa ggcc 2416 2095 DNA Human GP88 cDNA CDS (13)..(1791) Nucleotide sequence ofhuman granulin/epithelin precursor (human GP88). Human Granulin GenebankM75161. 16 cgcaggcaga cc atg tgg acc ctg gtg agc tgg gtg gcc tta aca gcaggg 51 Met Trp Thr Leu Val Ser Trp Val Ala Leu Thr Ala Gly 1 5 10 ctggtg gct gga acg cgg tgc cca gat ggt cag ttc tgc cct gtg gcc 99 Leu ValAla Gly Thr Arg Cys Pro Asp Gly Gln Phe Cys Pro Val Ala 15 20 25 tgc tgcctg gac ccc gga gga gcc agc tac agc tgc tgc cgt ccc ctt 147 Cys Cys LeuAsp Pro Gly Gly Ala Ser Tyr Ser Cys Cys Arg Pro Leu 30 35 40 45 ctg gacaaa tgg ccc aca aca ctg agc agg cat ctg ggt ggc ccc tgc 195 Leu Asp LysTrp Pro Thr Thr Leu Ser Arg His Leu Gly Gly Pro Cys 50 55 60 cag gtt gatgcc cac tgc tct gcc ggc cac tcc tgc atc ttt acc gtc 243 Gln Val Asp AlaHis Cys Ser Ala Gly His Ser Cys Ile Phe Thr Val 65 70 75 tca ggg act tccagt tgc tgc ccc ttc cca gag gcc gtg gca tgc ggg 291 Ser Gly Thr Ser SerCys Cys Pro Phe Pro Glu Ala Val Ala Cys Gly 80 85 90 gat ggc cat cac tgctgc cca cgg ggc ttc cac tgc agt gca gac ggg 339 Asp Gly His His Cys CysPro Arg Gly Phe His Cys Ser Ala Asp Gly 95 100 105 cga tcc tgc ttc caaaga tca ggt aac aac tcc gtg ggt gcc atc cag 387 Arg Ser Cys Phe Gln ArgSer Gly Asn Asn Ser Val Gly Ala Ile Gln 110 115 120 125 tgc cct gat agtcag ttc gaa tgc ccg gac ttc tcc acg tgc tgt gtt 435 Cys Pro Asp Ser GlnPhe Glu Cys Pro Asp Phe Ser Thr Cys Cys Val 130 135 140 atg gtc gat ggctcc tgg ggg tgc tgc ccc atg ccc cag gct tcc tgc 483 Met Val Asp Gly SerTrp Gly Cys Cys Pro Met Pro Gln Ala Ser Cys 145 150 155 tgt gaa gac agggtg cac tgc tgt ccg cac ggt gcc ttc tgc gac ctg 531 Cys Glu Asp Arg ValHis Cys Cys Pro His Gly Ala Phe Cys Asp Leu 160 165 170 gtt cac acc cgctgc atc aca ccc acg ggc acc cac ccc ctg gca aag 579 Val His Thr Arg CysIle Thr Pro Thr Gly Thr His Pro Leu Ala Lys 175 180 185 aag ctc cct gcccag agg act aac agg gca gtg gcc ttg tcc agc tcg 627 Lys Leu Pro Ala GlnArg Thr Asn Arg Ala Val Ala Leu Ser Ser Ser 190 195 200 205 gtc atg tgtccg gac gca cgg tcc cgg tgc cct gat ggt tct acc tgc 675 Val Met Cys ProAsp Ala Arg Ser Arg Cys Pro Asp Gly Ser Thr Cys 210 215 220 tgt gag ctgccc agt ggg aag tat ggc tgc tgc cca atg ccc aac gcc 723 Cys Glu Leu ProSer Gly Lys Tyr Gly Cys Cys Pro Met Pro Asn Ala 225 230 235 acc tgc tgctcc gat cac ctg cac tgc tgc ccc caa gac act gtg tgt 771 Thr Cys Cys SerAsp His Leu His Cys Cys Pro Gln Asp Thr Val Cys 240 245 250 gac ctg atccag agt aag tgc ctc tcc aag gag aac gct acc acg gac 819 Asp Leu Ile GlnSer Lys Cys Leu Ser Lys Glu Asn Ala Thr Thr Asp 255 260 265 ctc ctc actaag ctg cct gcg cac aca gtg ggc gat gtg aaa tgt gac 867 Leu Leu Thr LysLeu Pro Ala His Thr Val Gly Asp Val Lys Cys Asp 270 275 280 285 atg gaggtg agc tgc cca gat ggc tat acc tgc tgc cgt cta cag tcg 915 Met Glu ValSer Cys Pro Asp Gly Tyr Thr Cys Cys Arg Leu Gln Ser 290 295 300 ggg gcctgg ggc tgc tgc cct ttt acc cag gct gtg tgc tgt gag gac 963 Gly Ala TrpGly Cys Cys Pro Phe Thr Gln Ala Val Cys Cys Glu Asp 305 310 315 cac atacac tgc tgt ccc gcg ggg ttt acg tgt gac acg cag aag ggt 1011 His Ile HisCys Cys Pro Ala Gly Phe Thr Cys Asp Thr Gln Lys Gly 320 325 330 acc tgtgaa cag ggg ccc cac cag gtg ccc tgg atg gag aag gcc cca 1059 Thr Cys GluGln Gly Pro His Gln Val Pro Trp Met Glu Lys Ala Pro 335 340 345 gct cacctc agc ctg cca gac cca caa gcc ttg aag aga gat gtc ccc 1107 Ala His LeuSer Leu Pro Asp Pro Gln Ala Leu Lys Arg Asp Val Pro 350 355 360 365 tgtgat aat gtc agc agc tgt ccc tcc tcc gat acc tgc tgc caa ctc 1155 Cys AspAsn Val Ser Ser Cys Pro Ser Ser Asp Thr Cys Cys Gln Leu 370 375 380 acgtct ggg gag tgg ggc tgc tgt cca atc cca gag gct gtc tgc tgc 1203 Thr SerGly Glu Trp Gly Cys Cys Pro Ile Pro Glu Ala Val Cys Cys 385 390 395 tcggac cac cag cac tgc tgc ccc cag cga tac acg tgt gta gct gag 1251 Ser AspHis Gln His Cys Cys Pro Gln Arg Tyr Thr Cys Val Ala Glu 400 405 410 gggcag tgt cag cga gga agc gag atc gtg gct gga ctg gag aag atg 1299 Gly GlnCys Gln Arg Gly Ser Glu Ile Val Ala Gly Leu Glu Lys Met 415 420 425 cctgcc cgc cgc ggt tcc tta tcc cac ccc aga gac atc ggc tgt gac 1347 Pro AlaArg Arg Gly Ser Leu Ser His Pro Arg Asp Ile Gly Cys Asp 430 435 440 445cag cac acc agc tgc ccg gtg ggc gga acc tgc tgc ccg agc cag ggt 1395 GlnHis Thr Ser Cys Pro Val Gly Gly Thr Cys Cys Pro Ser Gln Gly 450 455 460ggg agc tgg gcc tgc tgc cag ttg ccc cat gct gtg tgc tgc gag gat 1443 GlySer Trp Ala Cys Cys Gln Leu Pro His Ala Val Cys Cys Glu Asp 465 470 475cgc cag cac tgc tgc ccg gct ggc tac acc tgc aac gtg aag gct cga 1491 ArgGln His Cys Cys Pro Ala Gly Tyr Thr Cys Asn Val Lys Ala Arg 480 485 490tcc tgc gag aag gaa gtg gtc tct gcc cag cct gcc acc ttc ctg gcc 1539 SerCys Glu Lys Glu Val Val Ser Ala Gln Pro Ala Thr Phe Leu Ala 495 500 505cgt agc cct cac gtg ggt gtg aag gac gtg gag tgt ggg gaa gga cac 1587 ArgSer Pro His Val Gly Val Lys Asp Val Glu Cys Gly Glu Gly His 510 515 520525 ttc tgc cat gat aac cag acc tgc tgc cga gac aac cga cag ggc tgg 1635Phe Cys His Asp Asn Gln Thr Cys Cys Arg Asp Asn Arg Gln Gly Trp 530 535540 gcc tgc tgt ccc tac gcc cag ggc gtc tgt tgt gct gat cgg cgc cac 1683Ala Cys Cys Pro Tyr Ala Gln Gly Val Cys Cys Ala Asp Arg Arg His 545 550555 tgc tgt cct gct ggc ttc cgc tgc gca cgc agg ggt acc aag tgt ttg 1731Cys Cys Pro Ala Gly Phe Arg Cys Ala Arg Arg Gly Thr Lys Cys Leu 560 565570 cgc agg gag gcc ccg cgc tgg gac gcc cct ttg agg gac cca gcc ttg 1779Arg Arg Glu Ala Pro Arg Trp Asp Ala Pro Leu Arg Asp Pro Ala Leu 575 580585 aga cag ctg ctg tgagggacag tactgaagac tctgcagccc tcgggacccc 1831 ArgGln Leu Leu 590 actcggaggg tgccctctgc tcaggcctcc ctagcacctc cccctaaccaaattctccct 1891 ggaccccatt ctgagctccc catcaccatg ggaggtgggg cctcaatctaaggcccttcc 1951 ctgtcagaag ggggttgagg caaaagccca ttacaagctg ccatcccctccccgtttcag 2011 tggaccctgt ggccaggtgc ttttccctat ccacaggggt gtttgtgtgttgggtgtgct 2071 ttcaataaag tttgtcactt tctt 2095 17 593 PRT Human GP88cDNA 17 Met Trp Thr Leu Val Ser Trp Val Ala Leu Thr Ala Gly Leu Val Ala1 5 10 15 Gly Thr Arg Cys Pro Asp Gly Gln Phe Cys Pro Val Ala Cys CysLeu 20 25 30 Asp Pro Gly Gly Ala Ser Tyr Ser Cys Cys Arg Pro Leu Leu AspLys 35 40 45 Trp Pro Thr Thr Leu Ser Arg His Leu Gly Gly Pro Cys Gln ValAsp 50 55 60 Ala His Cys Ser Ala Gly His Ser Cys Ile Phe Thr Val Ser GlyThr 65 70 75 80 Ser Ser Cys Cys Pro Phe Pro Glu Ala Val Ala Cys Gly AspGly His 85 90 95 His Cys Cys Pro Arg Gly Phe His Cys Ser Ala Asp Gly ArgSer Cys 100 105 110 Phe Gln Arg Ser Gly Asn Asn Ser Val Gly Ala Ile GlnCys Pro Asp 115 120 125 Ser Gln Phe Glu Cys Pro Asp Phe Ser Thr Cys CysVal Met Val Asp 130 135 140 Gly Ser Trp Gly Cys Cys Pro Met Pro Gln AlaSer Cys Cys Glu Asp 145 150 155 160 Arg Val His Cys Cys Pro His Gly AlaPhe Cys Asp Leu Val His Thr 165 170 175 Arg Cys Ile Thr Pro Thr Gly ThrHis Pro Leu Ala Lys Lys Leu Pro 180 185 190 Ala Gln Arg Thr Asn Arg AlaVal Ala Leu Ser Ser Ser Val Met Cys 195 200 205 Pro Asp Ala Arg Ser ArgCys Pro Asp Gly Ser Thr Cys Cys Glu Leu 210 215 220 Pro Ser Gly Lys TyrGly Cys Cys Pro Met Pro Asn Ala Thr Cys Cys 225 230 235 240 Ser Asp HisLeu His Cys Cys Pro Gln Asp Thr Val Cys Asp Leu Ile 245 250 255 Gln SerLys Cys Leu Ser Lys Glu Asn Ala Thr Thr Asp Leu Leu Thr 260 265 270 LysLeu Pro Ala His Thr Val Gly Asp Val Lys Cys Asp Met Glu Val 275 280 285Ser Cys Pro Asp Gly Tyr Thr Cys Cys Arg Leu Gln Ser Gly Ala Trp 290 295300 Gly Cys Cys Pro Phe Thr Gln Ala Val Cys Cys Glu Asp His Ile His 305310 315 320 Cys Cys Pro Ala Gly Phe Thr Cys Asp Thr Gln Lys Gly Thr CysGlu 325 330 335 Gln Gly Pro His Gln Val Pro Trp Met Glu Lys Ala Pro AlaHis Leu 340 345 350 Ser Leu Pro Asp Pro Gln Ala Leu Lys Arg Asp Val ProCys Asp Asn 355 360 365 Val Ser Ser Cys Pro Ser Ser Asp Thr Cys Cys GlnLeu Thr Ser Gly 370 375 380 Glu Trp Gly Cys Cys Pro Ile Pro Glu Ala ValCys Cys Ser Asp His 385 390 395 400 Gln His Cys Cys Pro Gln Arg Tyr ThrCys Val Ala Glu Gly Gln Cys 405 410 415 Gln Arg Gly Ser Glu Ile Val AlaGly Leu Glu Lys Met Pro Ala Arg 420 425 430 Arg Gly Ser Leu Ser His ProArg Asp Ile Gly Cys Asp Gln His Thr 435 440 445 Ser Cys Pro Val Gly GlyThr Cys Cys Pro Ser Gln Gly Gly Ser Trp 450 455 460 Ala Cys Cys Gln LeuPro His Ala Val Cys Cys Glu Asp Arg Gln His 465 470 475 480 Cys Cys ProAla Gly Tyr Thr Cys Asn Val Lys Ala Arg Ser Cys Glu 485 490 495 Lys GluVal Val Ser Ala Gln Pro Ala Thr Phe Leu Ala Arg Ser Pro 500 505 510 HisVal Gly Val Lys Asp Val Glu Cys Gly Glu Gly His Phe Cys His 515 520 525Asp Asn Gln Thr Cys Cys Arg Asp Asn Arg Gln Gly Trp Ala Cys Cys 530 535540 Pro Tyr Ala Gln Gly Val Cys Cys Ala Asp Arg Arg His Cys Cys Pro 545550 555 560 Ala Gly Phe Arg Cys Ala Arg Arg Gly Thr Lys Cys Leu Arg ArgGlu 565 570 575 Ala Pro Arg Trp Asp Ala Pro Leu Arg Asp Pro Ala Leu ArgGln Leu 580 585 590 Leu

What is claimed as new and desired to be protected by Letters Patent is:1. A pharmaceutical composition comprising a GP88 antagonizing agent anda pharmaceutically acceptable carrier.
 2. A composition according toclaim 1 wherein said agent is an anti-GP88 antibody.
 3. A compositionaccording to claim 2 wherein said antibody is a neutralizing antibody.4. A composition according to claim 3 wherein said antibody is madeagainst a GP88 sequence between aminoacid 334 and
 362. 5. A compositionaccording to claim 2 wherein said antibody is selected from the groupconsisting of anti-K19T, anti-S14R, anti-E19V and anti-A14R antibodies.6. A composition according to claim 1 wherein said agent is an antisenseoligonucleotide.
 7. A composition according to claim 6 wherein saidantisense oligonucleotide is an antisense RNA sequence having sufficientcomplementarity to GP88 mRNA to cause hybridization between saidantisense RNA and said GP88 mRNA and inhibition of the translation ofsaid GP88 mRNA.
 8. A composition according to claim 6 wherein saidantisense oligonucleotide is an antisense DNA sequence having sufficientcomplementarity to said GP88 mRNA to inhibit expression of said GP88. 9.A composition according to claim 1 wherein said agent is a vectorsuitable for expressing an antisense nucleotide sequence havingsufficient complementarity to GP88 mRNA to inhibit expression of GP88 incells.
 10. An expression vector containing cDNA to GP88 or a variant ofGP88 capable of expressing GP88 protein, or a functional derivative ofGP88 protein, substantially free of other mammalian proteins.
 11. Acomposition according to claim 1 wherein said agent is a reagent thatinhibits GP88 mRNA or protein expression.
 12. A composition according toclaim 1 wherein said agent is a reagent that inhibits GP88 proteinsecretion.
 13. A composition according to claim 1 wherein said agent isan antibody to GP88 receptor that can block GP88 function.
 14. Acomposition according to claim 1 wherein said agent is an antagonistthat inhibits GP88 binding to its receptor.
 15. A method for treatingdiseases associated with increased expression of GP88 comprising thesteps of administering an effective amount of a GP88 antagonizing agentwherein said agent inhibits the production or biological activity ofGP88.
 16. A method according to claim 15 wherein said disease is cancer.17. A method according to claim 15 wherein said agent is an antisenseoligonucleotide at least 20 nucleotides in length and having sufficientcomplementarity to GP88 DNA to inhibit GP88 expression.
 18. A methodaccording to claim 15 wherein said agent is antisense RNA.
 19. A methodaccording to claim 15 wherein said agent is an anti-GP88 antibody.
 20. Amethod for diagnosing tumorigenicity comprising the steps of measuringthe level of GP88 expression in tissue extracts or biological fluids;measuring the level of GP88 expression in corresponding normal orperipheral tissues; and determining whether the measured level of GP88expression in tissue extracts is higher than the level in correspondingnormal or peripheral tissues.
 21. A method according to claim 16 whereinsaid measuring step comprises measuring GP88 mRNA expression.
 22. Amethod according to claim 16 where said measuring step comprisesmeasuring GP protein expression.
 23. A method for diagnosing diseasesassociated with alteration in GP88 biological action comprising thesteps of measuring GP88 binding to its cell surface receptors ormeasuring GP88 receptors expression.
 24. A method for treating diseasesassociated with alteration of GP88 biological activity comprising thesteps of administering an effective amount of a GP88 antagonizing agentinhibits biological activity of GP88.
 25. A method according to claim 24wherein said agent is a neutralizing anti-GP88 antibody.
 26. A methodaccording to claim 24 wherein said agent is an antagonist of GP88binding to its receptor.
 27. A method according to claim 24 wherein saidagent is an anti-GP88 receptor antibody.